Ketol-acid reductoisomerase using nadh

ABSTRACT

Methods for the evolution of NADPH binding ketol-acid reductoisomerase enzymes to acquire NADH binding functionality are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from  Pseudomonas  that have undergone co-factor switching to bind NADH are described.

This application claims the benefit of U.S. Provisional Applications,61/015,346, filed Dec. 20, 2007, and 61/109,297, filed Oct. 29, 2008.

FIELD OF THE INVENTION

The invention relates to protein evolution. Specifically, ketol-acidreductoisomerase enzymes have been evolved to use the cofactor NADHinstead of NADPH.

BACKGROUND OF THE INVENTION

Ketol-acid reductoisomerase enzymes are ubiquitous in nature and areinvolved in the production of valine and isoleucine, pathways that mayaffect the biological synthesis of isobutanol. Isobutanol isspecifically produced from catabolism of L-valine as a by-product ofyeast fermentation. It is a component of “fusel oil” that forms as aresult of incomplete metabolism of amino acids by yeasts. After theamine group of L-valine is harvested as a nitrogen source, the resultingα-keto acid is decarboxylated and reduced to isobutanol by enzymes ofthe Ehrlich pathway (Dickinson, et al., J. Biol. Chem. 273, 25752-25756,1998).

Addition of exogenous L-valine to the fermentation increases the yieldof isobutanol, as described by Dickinson et al., supra, wherein it isreported that a yield of isobutanol of 3 g/L is obtained by providingL-valine at a concentration of 20 g/L in the fermentation. In addition,production of n-propanol, isobutanol and isoamylalcohol has been shownby calcium alginate immobilized cells of Zymomonas mobilis (Oaxaca, etal., Acta Biotechnol., 11, 523-532, 1991).

An increase in the yield of C3-C5 alcohols from carbohydrates was shownwhen amino acids leucine, isoleucine, and/or valine were added to thegrowth medium as the nitrogen source (WO 2005040392).

While methods described above indicate the potential of isobutanolproduction via biological means these methods are cost prohibitive forindustrial scale isobutanol production. The biosynthesis of isobutanoldirectly from sugars would be economically viable and would represent anadvance in the art. However, to date the only ketol-acidreductoisomerase (KARI) enzymes known are those that bind NADPH in itsnative form, reducing the energy efficiency of the pathway. A KARI thatwould bind NADH would be beneficial and enhance the productivity of theisobutanol biosynthetic pathway by capitalizing on the NADH produced bythe existing glycolytic and other metabolic pathways in most commonlyused microbial cells. The discovery of a KARI enzyme that can use NADHas a cofactor as opposed to NADPH would be an advance in the art.

The evolution of enzymes having specificity for the NADH cofactor asopposed to NADPH is known for some enzymes and is commonly referred toas “cofactor switching”. See for example Eppink, et al. J. Mol. Biol.,(1999), 292, 87-96, describing the switching of the cofactor specificityof strictly NADPH-dependent p-Hydroxybenzoate hydroxylase (PHBH) fromPseudomonas fluorescens by site-directed mutagenesis; and Nakanishi, etal., J. Biol. Chem., (1997), 272, 2218-2222, describing the use ofsite-directed mutagenesis on a mouse lung carbonyl reductase in whichThr-38 was replaced by Asp (T38D) resulting in an enzyme having a200-fold increase in the K_(m) values for NADP(H) and a correspondingdecrease of more than 7-fold in those for NAD(H). Co-factor switchinghas been applied to a variety of enzymes including monooxygenases,(Kamerbeek, et al., Eur. J, Biochem., (2004), 271, 2107-2116);dehydrogenases; Nishiyama, et al., J. Biol. Chem., (1993), 268,4656-4660; Ferredoxin-NADP reductase, Martinez-Julvez, et al., Biophys.Chem., (2005), 115, 219-224); and oxidoreductases (US2004/0248250).

Rane et al., (Arch. Biochem. Biophys., (1997), 338, 83-89) discusscofactor switching of a ketol acid reductoisomerase isolated from E.coli by targeting four residues in the enzyme for mutagenesis, (R68,K69, K75, and R76,); however the effectiveness of this method is indoubt.

Although the above cited methods suggest that it is generally possibleto switch the cofactor specificity between NADH and NADPH, the methodsare enzyme specific and the outcomes unpredictable. The development of aketol-acid reductoisomerase having a high specificity for NADH asopposed to NADPH would greatly enhance its effectiveness in theisobutanol biosynthetic pathway, however, no such KARI enzyme has beenreported.

Applicants have solved the stated problem by identifying a number ofmutant ketol-acid reductoisomerase enzymes that have a preference forbinding NADH as opposed to NADPH.

SUMMARY OF THE INVENTION

The invention relates to a method for the evolution of ketol-acidreductoisomerase (KARI) enzymes from binding the cofactor NADPH tobinding NADH. The method involves mutagenesis of certain specificresidues in them KARI enzyme to produce the co-factor switching.

Accordingly the invention provides a mutant ketol-acid reductoisomeraseenzyme comprising the amino acid sequence as set forth in SEQ ID NO: 29.

Alternatively the invention provides a mutant ketol-acidreductoisomerase enzyme having the amino acid sequence selected from thegroup consisting of SEQ ID NO: 19, 24, 25, 26, 27, 28, 67, 68, 69, and70.

In a preferred embodiment a mutant ketol-acid reductoisomerase enzyme isprovided as set forth in SEQ ID NO:17 comprising at least one mutationat a residue selected from the group consisting of 24, 33, 47, 50, 52,53, 61, 80, 115, 156, 165, and 170.

In a specific embodiment the invention provides a mutant ketol-acidreductoisomerase enzyme as set forth in SEQ ID NO:17 wherein:

-   -   a) the residue at position 47 has an amino acid substation        selected from the group consisting of A, C, D, F, G, I, L, N, P,        and Y;    -   b) the residue at position 50 has an amino acid substitution        selected from the group consisting of A, C, D, E, F, G, M, N, V,        W;    -   c) the residue at position 52 has an amino acid substitution        selected from the group consisting of A, C, D, G, H, N, S;    -   d) the residue at position 53 has an amino acid substitution        selected from the group consisting of A, H, I, W;    -   e) the residue at position 156 has an amino acid substitution of        V;    -   f) the residue at position 165 has an amino acid substitution of        M;    -   g) the residue at position 61 has an amino acid substitution of        F;    -   h) the residue at position 170 has an amino acid substitution of        A;    -   i) the residue at position 24 has an amino acid substitution of        F;    -   j) the residue at position 33 has an amino acid substitution of        L;    -   k) the residue at position 80 has an amino acid substitution of        1; and    -   l) the residue at position 115 has an amino acid substitution of        L.

In another embodiment the invention provides a method for the evolutionof a NADPH binding ketol-acid reductoisomerase enzyme to an NADH usingform comprising:

-   -   a) providing a ketol-acid reductoisomerase enzyme which uses        NADPH having a specific native amino acid sequence;    -   b) identifying the cofactor switching residues in the enzyme        of a) based on the amino acid sequence of the Pseudomonas        fluorescens ketol-acid reductoisomerase enzyme as set for the in        SEQ ID NO:17 wherein the cofactor switching residues are at        positions selected from the group consisting of; 24, 33, 47, 50,        52, 53, 61, 80, 115, 156, 165, and 170;    -   c) creating mutations in at least one of the cofactor switching        residues of b) to create a mutant enzyme wherein said mutant        enzyme binds NADH.

In an alternate embodiment the invention provides a method for theproduction of isobutanol comprising:

-   -   a) providing a recombinant microbial host cell comprising the        following genetic constructs:        -   i) at least one genetic construct encoding an acetolactate            synthase enzyme for the conversion of pyruvate to            acetolactate;        -   ii) at least one genetic construct encoding a mutant            ketol-acid reductoisomerase enzyme of the invention;        -   iii) at least one genetic construct encoding an acetohydroxy            acid dehydratase for the conversion of            2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step            c);        -   iv) at least one genetic construct encoding a branched-chain            keto acid decarboxylase, of the conversion of            α-ketoisovalerate to isobutyraldehyde, (pathway step d);        -   v) at least one genetic construct encoding a branched-chain            alcohol dehydrogenase for the conversion of isobutyraldehyde            to isobutanol (pathway step e); and    -   b) growing the host cell of (a) under conditions where        iso-butanol is produced.

BRIEF DESCRIPTION OF THE FIGURES SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, the Figures, and the accompanying sequence descriptions,which form part of this application.

FIG. 1—Shows four different isobutanol biosynthetic pathways. The stepslabeled “a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j” and “k”represent the substrate to product conversions described below.

FIG. 2—Multiple sequence alignment (MSA) of KARI enzymes from differentrecourses. (a) MSA among three NADPH-requiring KARI enzymes; (b) MSAamong PF5-KARI and other KARI enzymes, with promiscuous nucleotidespecificity, where, MMC5—is from Methanococcus maripaludis C5; MMS2—isfrom Methanococcus maripaludis S2; MNSB—is from Methanococcus vannielliiSB; ilv5—is from Saccharomyces cerevisiae ilv5; KARI-D1—is fromSulfolobus solfataricus P2 ilvC; KARI-D2—is from Pyrobaculum aerophilumP2ilvC; and KARI S1—is from Ralstonia solanacearum GMI1000 ivlC.

FIG. 3—Interaction of phosphate binding loop with NADPH based onhomology modeling.

FIG. 4: KARI activities of top performers from the library C usingcofactors NADH versus NADPH. Activity and standard deviation werederived from triple experiments. The mutation information is as follows:C3A7=R47Y/S50A/T52D/V53W; C3A10=R47Y/S50A/T52G/V53W;C3B11=R47F/S50A/T52D/V53W; C3C8=R47G/S50M/T52D/V53W; andC4D12=R47C/S50MT52D/V53W

FIG. 5—(a) KARI activities of top performers from libraries E, F and Gusing cofactors NADH versus NADPH. (b) KARI activities of positivecontrol versus wild type Pf5-ilvC using cofactors NADH. Activity andstandard deviation were derived from at least three parallelexperiments. “Wt” represents the wild type of Pf5-ilvC and “Neg” meansnegative control.

Experiments for NADH and NADPH reactions in (a) were 30 minutes; in (b)were 10 minutes.

FIG. 6—Activities of top performers from library H using cofactors NADHversus NADPH. Activity and standard deviation were derived from tripleexperiments. Mutation information is as follows: 24F9=R47P/S50G/T52D;68F10=R47P/T52S; 83G10=R47P/S50D/T52S; 39G4=R47P/S50C/T52D;91A9=R47P/S50CT52D; and C3B11=R47F/S50A/T52D/V53W

FIG. 7—Thermostability of PF5-ilvC. The remaining activity of the enzymeafter heating at certain temperatures for 10 min was the average numberof triple experiments and normalized to the activity measured at roomtemperature.

FIG. 8—Multiple sequence alignment among 5 naturally existing KARImolecules. The positions bolded and grey highlighted were identified byerror prone PCR and the positions only grey highlighted were targetedfor mutagenesis.

FIG. 9—Alignment of the twenty-four functionally verified KARIsequences. The GxGXX(G/A) motif involved in the binding of NAD(P)H isindicated below the alignment.

FIG. 10—An example of the alignment of Pseudomonas fluorescens Pf-5 KARIto the profile HMM of KARI. The eleven positions that are responsiblefor co-factor switching are bolded and shaded in grey.

Table 9—is a table of the Profile HMM of the KARI enzymes described inExample 5. The eleven positions in the profile HMM representing thecolumns in the alignment which correspond to the eleven cofactorswitching positions in Pseudomonas fluorescens Pf-5 KARI are identifiedas positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170. Thelines corresponding to these positions in the model file are highlightedin yellow. Table 9 is submitted herewith electronically and isincorporated herein by reference.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with the World Intellectual Property Organization (WIPO)Standard ST.25 (1998) and the sequence listing requirements of the EPOand PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 OLIGONUCLEOTIDE PRIMERS USED IN THIS INVENTION SEQUENCE ID No.SEQUENCE Description 1 TGATGAACATCTTCGCGTATTCGCCGTCCT Reverse Primer forpBAD vector 2 GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNN Forward primerGGCTNNCTGGGCCAAGGCT GAAGCCCACGGCTTG library C 3GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCTCG Forward primer forGCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG library E 4GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCNNT Forward primer forGCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG library F 5GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCTCG Forward primer forGCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG library G 6GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNNT Forward primer forGCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG library H 7 AAGATTAGCGGATCCTACCTSequencing primer (forward) 8 AACAGCCAAGCTTTTAGTTC Sequencing primer(reverse) 20 CTCTCTACTGTTTCTCCATACCCG pBAD_266-021308f 21CAAGCCGTGGGCTTCAGCCTTGGCKNN PF5_53Mt022908r 22 CGGTTTCAGTCTCGTCCTTGAAGpBAD_866-021308 49 GCTCAAGCANNKAACCTGAAGG pBAD-405- C33_090808f 50CCTTCAGGTTKNNTGCTTGAGC pBAD-427- C33_090808r 51 GTAGACGTGNNKGTTGGCCTGpBAD-435- T43_090808f 52 CAGGCCAACKNNCACGTCTAC pBAD-456- T43_090808r 53CTGAAGCCNNKGGCNNKAAAGTGAC pBAD-484- H59L61_090808f 54GTCACTTTKNNGCCKNNGGCTTCAG pBAD-509- H59L61_090808r 55GCAGCCGTTNNKGGTGCCGACT pBAD-519- A71_090808f 56 AGTCGGCACCKNNAACGGCTGCpBAD-541- A71_090808r 57 CATGATCCTGNNKCCGGAGGAG pBAD-545- T80_090808f 58CTCGTCCGGKNNCAGGATCATG pBAD-567- T80_090808r 59 CAAGAAGGGCNNKACTCTGGCCTpBAD-608- A101_090808f 60 AGGCCAGAGTKNNGCCCTTCTTG pBAD-631- A101_090808r61 GTTGTGCCTNNKGCCGACCTCG pBAD-663- R119_090808f 62CGAGGTCGGCKNNAGGCACAAC pBAD-685- R119_090808r

Additional sequences used in the application are listed below. Theabbreviated gene names in bracket are used in this disclosure.

SEQ ID NO: 9-Methanococcus maripaludis C5-ilvC (MMC5)—GenBank AccessionNumber NC_(—)009135.1 Region: 901034.902026SEQ ID NO: 10 is the Methanococcus maripaludis S2-ilvC (MMS2)—GenBankAccession Number NC_(—)005791.1 Region: 645729.646721SEQ ID NO: 11 is the Methanococcus vannielii SB-ilv5 (MVSB)—GenBankAccession Number NZ_AAWX01000002.1 Region: 302214.303206SEQ ID NO: 12 is the Saccharomyces cerevisiae ilv5 (ilv5)—GenBankAccession Number NC_(—)001144.4 Region: 838065.839252SEQ ID NO: 13 is the Sulfolobus solfataricus P2 ilvC (KARI-D1)—GenBankAccession Number NC_(—)002754.1 Region: 506253.507260SEQ ID NO: 14 is the Pyrobaculum aerophilum str. IM2 ilvC(KARI-D2)—GenBank Accession Number NC_(—)003364.1 Region:1976281.1977267SEQ ID NO: 15 is the Ralstonia solanacearum GMI1000 ilvC(KARI-S1)—GenBank Accession Number NC_(—)003295.1 Region:2248264.2249280SEQ ID NO: 16 is the Pseudomonas aeruginosa PAO1 ilvC—GenBank AccessionNumber NC_(—)002516 Region: 5272455.5273471SEQ ID NO: 17 is the Pseudomonas fluorescens PF5 ilvC—GenBank AccessionNumber NC_(—)004129 Region: 6017379.6018395SEQ ID NO: 18 is the Spinacia oleracea ilvC (Spinach-KARI)—GenBankAccession Number NC_(—)002516 Region: 1.2050.SEQ ID NO: 19 is the amino acid sequence of the mutant(Y24F/R47Y/S50A/T52D/V53A/L61F/G170A) of the ilvC native protein ofPseudomonas fluorescens.SEQ ID NO: 23 is the DNA SEQ of the mutant(Y24F/R47Y/S50A/T52D/V53A/L61F/G170A) of the ilvC native protein ofPseudomonas fluorescens.SEQ ID NO: 24 is the amino acid SEQ of the mutant ZB1(Y24F/R47Y/S50A/T52D/V53A/L61F/A156V)SEQ ID NO: 25 is the amino acid SEQ of the mutant ZF3(Y24F/C33L/R47Y/S50A/T52D/V53A/L61F)SEQ ID NO: 26 is the amino acid SEQ of the mutant ZF2(Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V)SEQ ID NO: 27 is the amino acid SEQ of the mutant ZB3(Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A)SEQ ID NO: 28 is the amino acid SEQ of the mutant Z4B8(C33L/R47Y/S50A/T52D/V53A/L61F/T801/A156V/G170A)SEQ ID NO: 29 is a consensus amino acid sequence comprising allexperimentally verified KARI point mutations as based on SEQ ID NO:17.SEQ ID NO: 30 is the amino acid sequence for KARI from Natronomonaspharaonis DSM 2160SEQ ID NO: 31 is the amino acid sequence for KARI from Bacillus subtilissubsp. subtilis str. 168SEQ ID NO: 32 is the amino acid sequence for KARI from Corynebacteriumglutamicum ATCC13032SEQ ID NO: 33 is the amino acid sequence for KARI from PhaeospirilummolischianumSEQ ID NO: 34 is the amino acid sequence for KARI from Zymomonas mobilissubsp. mobilis ZM4SEQ ID NO: 35 is the amino acid sequence for KARI Alkalilimnicolaehrlichei MLHE-1SEQ ID NO: 36 is the amino acid sequence for KARI from Campylobacterlari RM2100SEQ ID NO: 37 is the amino acid sequence for KARI from Marinobacteraquaeolei VT8SEQ ID NO: 38 is the amino acid sequence for KARI Psychrobacter arcticus273-4SEQ ID NO: 39 is the amino acid sequence for KARI from Hahellachejuensis KCTC2396SEQ ID NO: 40 is the amino acid sequence for KARI from Thiobacillusdenitrificans ATCC25259SEQ ID NO: 41 is the amino acid sequence for KARI from Azotobactervinelandii AVOPSEQ ID NO: 42 is the amino acid sequence for KARI from Pseudomonassyringae pv. syringae B728aSEQ ID NO: 43 is the amino acid sequence for KARI from Pseudomonassyringae pv. tomato str. DC3000SEQ ID NO: 44 is the amino acid sequence for KARI from Pseudomonasputida KT2440SEQ ID NO: 45 is the amino acid sequence for KARI from Pseudomonasentomophila L48SEQ ID NO: 46 is the amino acid sequence for KARI from Pseudomonasmendocina ympSEQ ID NO: 47 is the amino acid sequence for KARI from Bacillus cereusATCC10987 NP_(—)977840.1SEQ ID NO: 48 is the amino acid sequence for KARI from Bacillus cereusATCC10987 NP_(—)978252.1SEQ ID NO: 63 is the amino acid sequence for KARI from Escherichiacoli-GenBank Accession Number P05793SEQ ID NO: 64 is the amino acid sequence for KARI from Marine GammaProteobacterium HTCC2207-GenBank Accession Number ZP_(—)01224863.1SEQ ID NO: 65 is the amino acid sequence for KARI from Desulfuromonasacetoxidans—GenBank Accession Number ZP_(—)01313517.1SEQ ID NO: 66 is the amino acid sequence for KARI from Pisum sativum(Pea)—GenBank Accession Number 082043SEQ ID NO: 67 is the amino acid sequence for the mutant 3361 G8(C33L/R47Y/S50A/T52D/V53A/L61F/T801)SEQ ID NO: 68 is the amino acid sequence for the mutant 2H10(Y24F/C33L/R47Y/S50A/T52D/V531/L61F/T801/A156V)SEQ ID NO: 69 is the amino acid sequence for the mutant 1D2(Y24F/R47Y/S50A/T52D/V53A/L61F/T801/A156VSEQ ID NO: 70 is the amino acid sequence for the mutant 3F12(Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T801/A156V).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the generation of mutated KARI enzymesto use NADH as opposed to NADPH. These co-factor switched enzymesfunction more effectively in microbial systems designed to produceisobutanol. Isobutanol is an important industrial commodity chemicalwith a variety of applications, where its potential as a fuel or fueladditive is particularly significant. Although only a four-carbonalcohol, butanol has the energy content similar to that of gasoline andcan be blended with any fossil fuel. Isobutanol is favored as a fuel orfuel additive as it yields only CO₂ and little or no SO_(x) or NO_(x)when burned in the standard internal combustion engine. Additionallybutanol is less corrosive than ethanol, the most preferred fuel additiveto date.

The following definitions and abbreviations are to be use for theinterpretation of the claims and the specification.

The term “invention” or “present invention” as used herein is meant toapply generally to all embodiments of the invention as described in theclaims as presented or as later amended and supplemented, or in thespecification.

The term “isobutanol biosynthetic pathway” refers to the enzymaticpathway to produce isobutanol. Preferred isobutanol biosyntheticpathways are illustrated in FIG. 1 and described herein.

The term “NADPH consumption assay” refers to an enzyme assay for thedetermination of the specific activity of the KARI enzyme, involvingmeasuring the disappearance of the KARI cofactor, NADPH, from the enzymereaction.

“KARI” is the abbreviation for the enzyme Ketol-acid reductoisomerase.

The term “close proximity” when referring to the position of variousamino acid residues of a KARI enzyme with respect to the adenosyl2′-phosphate of NADPH means amino acids in the three-dimensional modelfor the structure of the enzyme that are within about 4.5 Å of thephosphorus atom of the adenosyl 2′-phosphate of NADPH bound to theenzyme.

The term “Ketol-acid reductoisomerase” (abbreviated “KARI”), and“Acetohydroxy acid isomeroreductase” will be used interchangeably andrefer the enzyme having the EC number, EC 1.1.1.86 (Enzyme Nomenclature1992, Academic Press, San Diego). Ketol-acid reductoisomerase catalyzesthe reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate, as morefully described below. These enzymes are available from a number ofsources, including, but not limited to E. coli GenBank Accession NumberNC-000913 REGION: 3955993.3957468, Vibrio cholerae GenBank AccessionNumber NC-002505 REGION: 157441.158925, Pseudomonas aeruginosa, GenBankAccession Number NC-002516, (SEQ ID NO: 16) REGION: 5272455.5273471, andPseudomonas fluorescens GenBank Accession Number NC-004129 (SEQ ID NO:17) REGION: 6017379.6018395. As used herein the term “Class I ketol-acidreductoisomerase enzyme” means the short form that typically has between330 and 340 amino acid residues, and is distinct from the long form,called class II, that typically has approximately 490 residues.

The term “acetolactate synthase” refers to an enzyme that catalyzes theconversion of pyruvate to acetolactate and CO₂. Acetolactate has twostereoisomers ((R)— and (S)—); the enzyme prefers the (S)— isomer, whichis made by biological systems. Preferred acetolactate synthases areknown by the EC number 2.2.1.6 9 (Enzyme Nomenclature 1992, AcademicPress, San Diego). These enzymes are available from a number of sources,including, but not limited to, Bacillus subtilis (GenBank Nos: CAB15618,Z99122, NCBI (National Center for Biotechnology Information) amino acidsequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae(GenBank Nos: AAA25079 (SEQ ID NO:2), M73842 (SEQ ID NO:1)), andLactococcus lactis (GenBank Nos: AAA25161, L16975).

The term “acetohydroxy acid dehydratase” refers to an enzyme thatcatalyzes the conversion of 2,3-dihydroxy-isovalerate toα-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known bythe EC number 4.2.1.9. These enzymes are available from a vast array ofmicroorganisms, including, but not limited to, E. coli (GenBank Nos:YP_(—)026248, NC_(—)000913, S. cerevisiae (GenBank Nos: NP_(—)012550,NC_(—)001142), M. maripaludis (GenBank Nos: CAF29874, BX957219), and B.subtilis (GenBank Nos: CAB14105, Z99115).

The term “branched-chain α-keto acid decarboxylase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyraldehydeand CO₂. Preferred branched-chain α-keto acid decarboxylases are knownby the EC number 4.1.1.72 and are available from a number of sources,including, but not limited to, Lactococcus lactis (GenBank Nos:AAS49166, AY548760; CAG34226, AJ746364, Salmonella typhimurium (GenBankNos: NP-461346, NC-003197), and Clostridium acetobutylicum (GenBank Nos:NP-149189, NC-001988).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyraldehyde to isobutanol. Preferredbranched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but may also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH(reduced nicotinamide adenine dinucleotide) and/or NADPH as electrondonor and are available from a number of sources, including, but notlimited to, S. cerevisiae (GenBank Nos: NP-010656, NC-001136; NP-014051,NC-001145), E. coli (GenBank Nos: NP-417-484, and C. acetobutylicum(GenBank Nos: NP-349892, NC_(—)003030).

The term “branched-chain keto acid dehydrogenase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA(isobutyryl-cofactor A), using NAD⁺ (nicotinamide adenine dinucleotide)as electron acceptor. Preferred branched-chain keto acid dehydrogenasesare known by the EC number 1.2.4.4. These branched-chain keto aciddehydrogenases comprise four subunits, and sequences from all subunitsare available from a vast array of microorganisms, including, but notlimited to, B. subtilis (GenBank Nos: CAB14336, Z99116; CAB14335,Z99116; CAB14334, Z99116; and CAB14337, Z99116) and Pseudomonas putida(GenBank Nos: AAA65614, M57613; AAA65615, M57613; AAA65617, M57613; andAAA65618, M57613).

The terms “k_(cat)” and “K_(m)” are known to those skilled in the artand are described in Enzyme Structure and Mechanism, 2^(nd) ed. (Ferst;W.H. Freeman: NY, 1985; pp 98-120). The term “k_(cat)”, often called the“turnover number”, is defined as the maximum number of substratemolecules converted to products per active site per unit time, or thenumber of times the enzyme turns over per unit time. k_(cat)=Vmax/[E],where [E] is the enzyme concentration (Ferst, supra). The terms “totalturnover” and “total turnover number” are used herein to refer to theamount of product formed by the reaction of a KARI enzyme withsubstrate.

The term “catalytic efficiency” is defined as the k_(cat)/K_(M) of anenzyme. Catalytic efficiency is used to quantify the specificity of anenzyme for a substrate.

The term “isolated nucleic acid molecule”, “isolated nucleic acidfragment” and “genetic construct” will be used interchangeably and willmean a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HLeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

The term “Gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

As used herein the term “Coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

The term “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into the genome of a host organism, resulting ingenetically stable inheritance. Host organisms containing thetransformed nucleic acid fragments are referred to as “transgenic” or“recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein the term “Codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withouteffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook et al. (Sambrook,Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989) (hereinafter “Maniatis”); and by Silhavy et al. (Experiments withGene Fusions, Cold Spring Harbor Laboratory Press Cold Spring Harbor,N.Y., 1984); and by Ausubel, F. M. et al., (Current Protocols inMolecular Biology, published by Greene Publishing Assoc. andWiley-Interscience, 1987).

The present invention addresses a need that arises in the microbialproduction of isobutanol where the ketol-acid reductoisomerase enzymeperforms a vital role. Wild type ketol-acid reductoisomerase enzymestypically use NADPH as their cofactor. However, in the formation ofisobutanol an excess of NADH is produced by ancillary metabolicpathways. The invention provides mutant Class I KARI enzymes that havebeen evolved to utilize NADH as a cofactor, overcoming the cofactorproblem and increasing the efficiency of the isobutanol biosyntheticpathway.

Production of isobutanol utilizes the glycolysis pathway present in thehost organism. During the production of two molecules of pyruvate fromglucose during glycolysis, there is net production of two molecules ofNADH from NAD⁺ by the glyceraldehyde-3-phosphate dehydrogenase reaction.During the further production of one molecule of isobutanol from twomolecules of pyruvate, there is net consumption of one molecule ofNADPH, by the KARI reaction, and one molecule of NADH by the isobutanoldehydrogenase reaction. The overall reaction of glucose to isobutanolthus leads to net production of one molecule of NADH and net consumptionof one molecule of NADPH. The interconversion of NADH with NADPH isgenerally slow and inefficient; thus, the NADPH consumed is generated bymetabolism (for example, by the pentose phosphate pathway) consumingsubstrate in the process. Meanwhile, the cell strives to maintainhomeostasis in the NAD⁺/NADH ratio, leading to the excess NADH producedin isobutanol production being consumed in wasteful reduction of othermetabolic intermediates; e.g., by the production of lactate frompyruvate. Thus, the imbalance between NADH produced and NADPH consumedby the isobutanol pathway leads to a reduction in the molar yield ofisobutanol produced from glucose in two ways: 1) unnecessary operationof metabolism to produce NADPH, and 2) wasteful reaction of metabolicintermediates to maintain NAD⁺/NADH homeostasis. The solution to thisproblem is to invent a KARI that is specific for NADH as its cofactor,so that both molecules of NADH produced in glycolysis are consumed inthe synthesis of isobutanol from pyruvate.

Keto Acid Reductoisomerase (KARI) Enzymes

Acetohydroxy acid isomeroreductase or Ketol-acid reductoisomerase (KARI;EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chainamino acids and is a key enzyme in their biosynthesis. KARI is found ina variety of organisms and amino acid sequence comparisons acrossspecies have revealed that there are 2 types of this enzyme: a shortform (class I) found in fungi and most bacteria, and a long form (classII) typical of plants.

Class I KARIs typically have between 330-340 amino acid residues. Thelong form KARI enzymes have about 490 amino acid residues. However, somebacteria such as Escherichia Coli possess a long form, where the aminoacid sequence differs appreciably from that found in plants. KARI isencoded by the ilvC gene and is an essential enzyme for growth of E.Coli and other bacteria in a minimal medium. Typically KARI uses NADPHas cofactor and requires a divalent cation such as Mg⁺⁺ for itsactivity. In addition to utilizing acetolactate in the valine pathway,KARI also converts acetohydroxybutanoate to dihydroxymethylpentanoate inthe isoleucine production pathway.

Class II KARIs generally consist of a 225-residue N-terminal domain anda 287-residue C-terminal domain. The N-terminal domain, which containsthe NADPH-binding site, has an α/β structure and resembles domains foundin other pyridine nucleotide-dependent oxidoreductases. The C-terminaldomain consists almost entirely of α-helices and is of a previouslyunknown topology.

The crystal structure of the E. Coli KARI enzyme at 2.6 Å resolution hasbeen solved (Tyagi, et al., Protein Science, 14, 3089-3100, 2005). Thisenzyme consists of two domains, one with mixed α/β structure which issimilar to that found in other pyridine nucleotide-dependentdehydrogenases. The second domain is mainly α-helical and shows strongevidence of internal duplication. Comparison of the active sites of KARIof E. Coli, Pseudomonas aeruginosa, and spinach showed that mostresidues in the active site of the enzyme occupy conserved positions.While the E. Coli KARI was crystallized as a tetramer, which is probablythe likely biologically active unit, the P. aeruginosa KARI (Ahn, etal., J. Mol. Biol., 328, 505-515, 2003) formed a dodecamer, and theenzyme from spinach formed a dimer. Known KARIs are slow enzymes with areported turnover number (k_(cat)) of 2 s⁻¹ (Aulabaugh et al.;Biochemistry, 29, 2824-2830, 1990) or 0.12 s⁻¹ (Rane et al., Arch.Biochem. Biophys. 338, 83-89, 1997) for acetolactate. Studies have shownthat genetic control of isoleucine-valine biosynthesis in E. coli isdifferent than that in Ps. aeruginosa (Marinus, et al., Genetics, 63,547-56, 1969).

Identification of Amino Acid Target Sites for Cofactor Switching

It was reported that phosphate p2′ oxygen atoms of NADPH form hydrogenbonds with side chains of Arg162, Ser165 and Ser167 of spinach KARI(Biou V. et al. The EMBO Journal, 16: 3405-3415, 1997). Multiplesequence alignments were performed, using vector NTI (Invitrogen Corp.Carlsbad, Calif.), with KARI enzymes from spinach, Pseudomonasaeruginosa (PAO-KARI) and Pseudomonas fluorescens (PF5-KARI). The NADPHbinding sites are shown in FIG. 2 a. The amino acids, argenine,threonine and serine appear to play similar roles in forming hydrogenbonds with phosphate p2′ oxygen atoms of NADPH in KARI enzymes. Studiesby Ahn et al (J. Mol. Biol., 328: 505-515, 2003) had identified threeNADPH phosphate binding sites (Arg47, Ser50 and Thr52) for Pseudomonasaeruginosa (PAO-KARI) following comparing its structure with that of thespinach KARI. Hypothesizing that these three NADPH phosphate bindingsites of the three KARI enzymes used in the disclosure were conserved,Arg47, Ser50 and Thr52 of PF5-KARI were targeted as the phosphatebinding sites for this enzyme. This hypothesis was further confirmedthrough homology modeling.

Multiple sequence alignment among PF5-ilvC and several other KARIenzymes with promiscuous nucleotide specificity was also performed. Asshown in FIG. 2 b, the amino acids of glycine (G50) and tryptophan(W53), in other KARI enzymes in FIG. 2 b, always appear together as apair in the sequences of those enzymes. It was therefore assumed thatthe tryptophan 53 bulky residue was important in determining nucleotidespecificity by reducing the size of nucleotide binding pocket to favorthe smaller nucleotide, NADH. Position 53 of PF5-ilvC was thereforechosen as a target for mutagenesis.

Several site-saturation gene libraries were prepared containing genesencoding KARI enzymes by commercially available kits for the generationof mutants. Clones from each library were screened for improved KARIactivity using the NADH consumption assay described herein. Screeningresulted in the identification of a number of genes having mutationsthat can be correlated to KARI activity. The location of the mutationswere identified using the amino acid sequence the Pseudomonasfluorescens PF5 ilvC protein (SEQ ID NO:17). Mutants having improvedKARI activity were those which had mutations at the following positions:47, 50, 52 and 53. More specifically desirable mutations included thefollowing substitutions:

-   -   a) the residue at position 47 has an amino acid substation        selected from the group consisting of A, C, D, F, G, I, L, N, P,        and Y;    -   b) the residue at position 50 has an amino acid substitution        selected from the group consisting of A, C, D, E, F, G, M, N, V,        W;    -   c) the residue at position 52 has an amino acid substitution        selected from the group consisting of A, C, D, G, H, N, S;    -   d) the residue at position 53 has an amino acid substitution        selected from the group consisting of A, H, I, W;

In another embodiment, additional mutagenesis, using error prone PCR,performed on the mutants listed above identified suitable mutationpositions as: 156, 165, 61, 170, 115 and 24. More specifically thedesirable mutants with lower K_(m) for NADH contained the followingsubstitutions:

-   -   e) the residue at position 156 has an amino acid substitution of        V;    -   f) the residue at position 165 has an amino acid substitution of        M;    -   g) the residue at position 61 has an amino acid substitution of        F;    -   h) the residue at position 170 has an amino acid substitution of        A;    -   i) the residue at position 24 has an amino acid substitution of        F; and    -   j) the residue at position 115 has an amino acid substitution of        L.

In further work, multiple sequence alignment of Pseudomonas fluorescensPF5-ilvC and Bacillus cereus ilvC1 and livC2 and spinach KARI wasperformed which allowed identification of positions 24, 33, 47, 50, 52,53, 61, 80, 156 and 170 for further mutagenesis. More specificallymutants with much lower K_(m) for NADH were obtained. These mutationsare also based on the Pseudomonas fluorescens, KARI enzyme (SEQ IDNO:17) as a reference sequence wherein the reference sequence comprisesat least one amino acid substitution selected from the group consistingof:

-   -   k) the residue at position 24 has an amino acid substitution of        phenylalanine;    -   l) the residue at position 50 has an amino acid substitution of        alanine;    -   m) the residue at position 52 has an amino acid substitution of        aspartic acid;    -   n) the residue at position 53 has an amino acid substitution of        alanine;    -   o) the residue at position 61 has an amino acid substitution of        phenylalanine;    -   p) the residue at position 156 has an amino acid substitution of        valine;    -   q) the residue at position 33 has an amino acid substitution of        leucine;    -   r) the residue at position 47 has an amino acid substitution of        tyrosine;    -   s) the residue at position 80 has an amino acid substitution of        isoleucine; and    -   t) the residue at position 170 has an amino acid substitution of        alanine.

The present invention includes a mutant polypeptide having KARIactivity, said polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 24, 25, 26, 27 and 28.

A consensus sequence for the mutant ilvC was generated from the multiplesequence alignment and is provided as SEQ ID NO: 29 which represents allexperimentally verified mutations of the KARI enzyme based on the aminoacid sequence of the KARI enzyme isolated from Pseudomonas fluorescens,(SEQ ID NO:17)

Additionally the present invention describes mutation positionsidentified using a profile Hidden Markov Model (HMM) built based onsequences of 25 functionally verified Class I and Class II KARI enzymes.Profile HMM identified mutation positions 24, 33, 47, 50, 52, 53, 61,80, 115, 156, and 170 (the numbering is based on the sequences ofPseudomonas fluorescens PF5 KARI). Thus, it will be appreciated by theskilled person that mutations at these positions, as well as thosediscussed above that have been experimentally verified will also giverise to KARI enzymes having the ability to bind NADH.

The Host Strains for Kari Engineering

Two host strains, E. coli TOP10 from Invitrogen and E. Coli Bw25113(ΔilvC, an ilvC gene-knockout), were used for making constructsover-expressing the KARI enzyme in this disclosure. In the Bw25113strain, the entire ilvC gene of the E. Coli chromosome was replaced by aKanamycin cassette using the Lambda red homology recombinationtechnology described by Kirill et al., (Kirill A. Datsenko and Barry L.Wanner, Proc. Natl. Acad. Sci. USA, 97, 6640-6645, 2000).

Homology Modeling of Pf5 Kari with Bound Substrates

The structure of PF5-KARI with bound NADPH, acetolactate and magnesiumions was built based on the crystal structure of P. aeruginosa PAO1-KARI(PDB ID 1 NP3, Ahn H. J. et al, J. Mol. Biol., 328, 505-515, 2003) whichhas 92% amino acid sequence homology to PF5 KARI. PAO1-KARI structure isa homo-dodecamer and each dodecamer consists of six homo-dimers withextensive dimer interface. The active site of KARI is located in thisdimer interface. The biological assembly is formed by six homo-dimerspositioned on the edges of a tetrahedron resulting in a highlysymmetrical dodecamer of 23 point group symmetry. For simplicity, onlythe dimeric unit (monomer A and monomer B) was built for the homologymodel of PF5-KARI in this study because the active site is in thehomo-dimer interface.

The model of PF5-KARI dimer was built based on the coordinates ofmonomer A and monomer B of PAO1-KARI and sequence of PF5-KARI usingDeepView/Swiss PDB viewer (Guex, N. and Peitsch, M. C. Electrophoresis18: 2714-2723, 1997). This model was then imported to program 0 (Jones,T. A. et al, Acta Crystallogr. A 47,110-119, 1991) on a Silicon Graphicssystem for further modification.

The structure of PAO1-KARI has no NADPH, substrate or inhibitor ormagnesium in the active site. Therefore, the spinach KARI structure (PDBID 1yve, Biou V. et al. The EMBO Journal, 16: 3405-3415, 1997.), whichhas magnesium ions, NADPH and inhibitor (N-Hydroxy-N-isopropyloxamate)in the acetolacate binding site, was used to model these molecules inthe active site. The plant KARI has very little sequence homology toeither PF5- or PAO1 KARI (<20% amino acid identity), however thestructures in the active site region of these two KARI enzymes are verysimilar. To overlay the active site of these two KARI structures,commands LSQ_ext, LSQ_improve, LSQ_mol in the program O were used toline up the active site of monomer A of spinach KARI to the monomer A ofPF5 KARI model. The coordinates of NADPH, two magnesium ions and theinhibitor bound in the active site of spinach KARI were extracted andincorporated to molecule A of PF5 KARI. A set of the coordinates ofthese molecules were generated for monomer B of PF5 KARI by applying thetransformation operator from monomer A to monomer B calculated by theprogram.

Because there is no NADPH in the active site of PAO1 KARI crystalstructure, the structures of the phosphate binding loop region in theNADPH binding site (residues 44-45 in PAO1 KARI, 157-170 in spinachKARI) are very different between the two. To model the NADPH bound form,the model of the PF5-KARI phosphate binding loop (44-55) was replaced bythat of 1yve (157-170). Any discrepancy of side chains between these twowas converted to those in the PF5-KARI sequence using the mutate_replacecommand in program 0, and the conformations of the replaced side-chainswere manually adjusted. The entire NADPH/Mg/inhibitor bound dimericPF5-KARI model went through one round of energy minimization usingprogram CNX (ACCELRYS San Diego Calif., Burnger, A. T. and Warren, G.L., Acta Crystallogr., D 54, 905-921, 1998) after which the inhibitorwas replaced by the substrate, acetolactate (AL), in the model. Theconformation of AL was manually adjusted to favor hydride transfer of C4of the nicotinamine of NADPH and the substrate. No further energyminimization was performed on this model (Coordinates of the modelcreated for this study are attached in a separate word file.). Theresidues in the phosphate binding loop and their interactions with NADPHare illustrated in FIG. 3.

Application of a Profile Hidden Markov Model for Identification ofResidue Positions Involved in Cofactor Switching in Kari Enzymes

Applicants have developed a method for identifying KARI enzymes and theresidue positions that are involved in cofactor switching from NADPH toNADH. To structurally characterize KARI enzymes, a Profile Hidden MarkovModel (HMM) was prepared as described in Example 5 using amino acidsequences of 25 KARI proteins with experimentally verified function asoutlined in Table 6. These KARIs were from [Pseudomonas fluorescens Pf-5(SEQ ID NO: 17), Sulfolobus solfataricus P2 (SEQ ID NO: 13), Pyrobaculumaerophilum str. IM2 (SEQ ID NO: 14), Natronomonas pharaonis DSM 2160(SEQ ID NO: 30), Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO:31), Corynebacterium glutamicum ATCC 13032 (SEQ ID NO: 32),Phaeospririlum molischianum (SEQ ID NO: 33), Ralstonia solanacearumGMI1000 (SEQ ID NO: 15), Zymomonas mobilis subsp. mobilis ZM4 (SEQ IDNO: 34), Alkalilimnicola ehrlichei MLHE-1 (SEQ ID NO: 35), Campylobacterlari RM2100 (SEQ ID NO: 36), Marinobacter aquaeolei VT8 (SEQ ID NO: 37),Psychrobacter arcticus 273-4 (SEQ ID NO: 38), Hahella chejuensis KCTC2396 (SEQ ID NO: 39), Thiobacillus denitrificans ATCC 25259 (SEQ ID NO:40), Azotobacter vinelandii AVOP (SEQ ID NO: 41), Pseudomonas syringaepv. syringae B728a (SEQ ID NO: 42), Pseudomonas syringae pv. tomato str.DC3000 (SEQ ID NO: 43), Pseudomonas putida KT2440 (Protein SEQ ID NO:44), Pseudomonas entomophila L48 (SEQ ID NO: 45), Pseudomonas mendocinaymp (SEQ ID NO: 46), Pseudomonas aeruginosa PAO1 (SEQ ID NO: 16),Bacillus cereus ATCC 10987 (SEQ ID NO: 47), Bacillus cereus ATCC 10987(SEQ ID NO: 48), and Spinacia oleracea (SEQ ID NO: 18).

In addition using methods disclosed in this application, sequences ofClass II KARI enzymes such as E. coli (SEQ ID NO: 63-GenBank AccessionNumber P05793), marine gamma Proteobacterium HTCC2207 (SEQ ID NO:64-GenBank Accession Number ZP_(—)01224863.1), Desulfuromonasacetoxidans (SEQ ID NO: 65-GenBank Accession Number ZP_(—)01313517.1)and Pisum sativum (pea) (SEQ ID NO: 66-GenBank Accession Number 082043)could be mentioned.

This Profile HMM for KARIs can be used to identify any KARI relatedproteins. Any protein that matches the Profile HMM with an E value of<10⁻³ using hmmsearch program in the HMMER package is expected to be afunctional KARI, which can be either a Class I and Class II KARI.Sequences matching the Profile HMM given herein are then analyzed forthe location of the 12 positions in Pseudomonas fluorescens Pf-5 thatswitches the cofactor from NADPH to NADH. The eleven nodes, as definedin the section of Profile HMM building, in the profile HMM representingthe columns in the alignment which correspond to the eleven co-factorswitching positions in Pseudomonas fluorescens Pf-5 KARI are identifiedas node 24, 33, 47, 50, 52, 53, 61, 80, 115, 156 and 170. The linescorresponding to these nodes in the model file are identified in Table9. One skilled in the art will readily be able to identify these 12positions in the amino acid sequence of a KARI protein from thealignment of the sequence to the profile HMM using hmmsearch program inHMMER package.

The KARI enzymes identified by this method, include both Class I andClass II KARI enzymes from either microbial or plant natural sources.Any KARI identified by this method may be used for heterologousexpression in microbial cells.

For example each of the KARI encoding nucleic acid fragments describedherein may be used to isolate genes encoding homologous proteins.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to: 1) methods of nucleic acid hybridization; 2) methodsof DNA and RNA amplification, as exemplified by various uses of nucleicacid amplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

Although the sequence homology between Class I and Class II KARI enzymesis low, the three dimensional structure of both Classes of the enzymes,particularly around the active site and nucleotide binding domains ishighly conserved (Tygai, R., et al., Protein Science, 34: 399-408,2001). The key amino acid residues that make up the substrate bindingpocket are highly conserved between these two Classes even though theymay not align well in a simple sequence comparison. It can therefore beconcluded that the residues affecting cofactor specificity identified inClass I KARI (e.g., positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156,and 170 of PF5 KARI) can be extended to Class II KARI enzymes.

Isobutanol Biosynthetic Pathways

Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas(EMP) pathway, the Entner and Doudoroff pathway and the pentosephosphate cycle as the central, metabolic routes to provide energy andcellular precursors for growth and maintenance. These pathways have incommon the intermediate glyceraldehyde-3-phosphate and, ultimately,pyruvate is formed directly or in combination with the EMP pathway.Subsequently, pyruvate is transformed to acetyl-cofactor A (acetyl-CoA)via a variety of means. Acetyl-CoA serves as a key intermediate, forexample, in generating fatty acids, amino acids and secondarymetabolites. The combined reactions of sugar conversion to pyruvateproduce energy (e.g. adenosine-5′-triphosphate, ATP) and reducingequivalents (e.g. reduced nicotinamide adenine dinucleotide, NADH, andreduced nicotinamide adenine dinucleotide phosphate, NADPH). NADH andNADPH must be recycled to their oxidized forms (NAD⁺ and NADP⁺,respectively). In the presence of inorganic electron acceptors (e.g. O₂,NO₃ ⁻ and SO₄ ²⁻), the reducing equivalents may be used to augment theenergy pool; alternatively, a reduced carbon byproduct may be formed.

There are four potential pathways for production of isobutanol fromcarbohydrate sources with recombinant microorganisms as shown in FIG. 1.All potential pathways for conversion of carbohydrates to isobutanolhave been described in the commonly owned U.S. patent application Ser.No. 11/586,315, which is incorporated herein by reference.

The preferred pathway for conversion of pyruvate to isobutanol consistsof enzymatic steps “a”, “b”, “c”, “d”, and “e” (FIG. 1) and includes thefollowing substrate to product conversions:

-   -   a) pyruvate to acetolactate, as catalyzed for example by        acetolactate synthase,    -   b) (S)-acetolactate to 2,3-dihydroxyisovalerate, as catalyzed        for example by acetohydroxy acid isomeroreductase,    -   c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed        for example by acetohydroxy acid dehydratase,    -   d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for        example by a branched-chain keto acid decarboxylase, and    -   e) isobutyraldehyde to isobutanol, as catalyzed for example by,        a branched-chain alcohol dehydrogenase.

This pathway combines enzymes involved in well-characterized pathwaysfor valine biosynthesis (pyruvate to α-ketoisovalerate) and valinecatabolism α-ketoisovalerate to isobutanol). Since many valinebiosynthetic enzymes also catalyze analogous reactions in the isoleucinebiosynthetic pathway, substrate specificity is a major consideration inselecting the gene sources. For this reason, the primary genes ofinterest for the acetolactate synthase enzyme are those from Bacillus(alsS) and Klebsiella (budB). These particular acetolactate synthasesare known to participate in butanediol fermentation in these organismsand show increased affinity for pyruvate over ketobutyrate (Gollop etal., J. Bacteriol. 172, 3444-3449, 1990); and (Holtzclaw et al., J.Bacteriol. 121, 917-922, 1975). The second and third pathway steps arecatalyzed by acetohydroxy acid reductoisomerase and dehydratase,respectively. These enzymes have been characterized from a number ofsources, such as for example, E. coli (Chunduru et al., Biochemistry 28,486-493, 1989); and (Flint et al., J. Biol. Chem. 268, 14732-14742,1993). The final two steps of the preferred isobutanol pathway are knownto occur in yeast, which can use valine as a nitrogen source and, in theprocess, secrete isobutanol. α-Ketoiso-valerate can be converted toisobutyraldehyde by a number of keto acid decarboxylase enzymes, such asfor example pyruvate decarboxylase. To prevent misdirection of pyruvateaway from isobutanol production, a decarboxylase with decreased affinityfor pyruvate is desired. So far, there are two such enzymes known in theart (Smit et al., Appl. Environ. Microbiol., 71, 303-311, 2005); and (dela Plaza et al., FEMS Microbiol. Lett., 238, 367-374, 2004). Bothenzymes are from strains of Lactococcus lactis and have a 50-200-foldpreference for ketoisovalerate over pyruvate. Finally, a number ofaldehyde reductases have been identified in yeast, many with overlappingsubstrate specificity. Those known to prefer branched-chain substratesover acetaldehyde include, but are not limited to, alcohol dehydrogenaseVI (ADH6) and Ypr1p (Larroy et al., Biochem. J. 361, 163-172, 2002); and(Ford et al., Yeast 19, 1087-1096, 2002), both of which use NADPH aselectron donor. An NADPH-dependent reductase, YqhD, active withbranched-chain substrates has also been recently identified in E. coli(Sulzenbacher et al., J. Mol. Biol. 342, 489-502, 2004).

Two of the other potential pathways for isobutanol production alsocontain the initial three steps of “a”, “b” and “c” (FIG. 1). Onepathway consists of enzymatic steps “a”, “b”, “c”, “f”, “g”, “e” (FIG.1). Step “f” containing a “branched-chain keto acid dehydrogenase” withan EC number 1.2.4.4. Step “g” containing an “acylating aldehydedehydrogenase” with a EC numbers 1.2.1.10 and 1.2.1.57 in addition tostep “e” containing the “branched chain alcohol dehydrogenase”. Theother potential pathway consists of steps “a”, “b”, “c”, “h”, “,”, (FIG.1). The term “transaminase” (step “h”) EC numbers 2.6.1.42 and 2.6.1.66.Step “h” consists of either a “valine dehydrogenase” with EC numbers1.4.1.8 and 1.4.1.9 or step “i”, a “valine decarboxylase” with an ECnumber 4.1.1.14. Finally step “j” will use an “omega transaminase” withan EC number 2.6.1.18 to generate isobutyraldehyde which will be reducedby step “e” to produce isobutanol. All potential pathways for conversionof pyruvate to isobutanol are depicted in FIG. 1.

Additionally, a number of organisms are known to produce butyrate and/orbutanol via a butyryl-CoA intermediate (Dürre, et al., FEMS Microbiol.Rev. 17, 251-262, 1995); and (Abbad-Andaloussi et al., Microbiology 142,1149-1158, 1996). Therefore isobutanol production in these organismswill take place using steps “k”, “g” and “e” shown in FIG. 1. Step “k”will use an “isobutyryl-CoA mutase” with an EC number 5.4.99.13. Thenest step will involve using the “acylating aldehyde dehydrogenase” withthe EC numbers 1.2.1.10 and 1.2.1.57 to produce isobutyraldehydefollowed by enzymatic step “e” to produce isobutanol. All these pathwaysare fully described in the commonly owned patent application CL3243Herein incorporated by reference.

Thus, in providing multiple recombinant pathways from pyruvate toisobutanol, there exist a number of choices to fulfill the individualconversion steps, and the person of skill in the art will be able toutilize publicly available sequences to construct the relevant pathways.

Microbial Hosts for Isobutanol Production

Microbial hosts for isobutanol production may be selected from bacteria,cyanobacteria, filamentous fungi and yeasts. The microbial host used forisobutanol production should be tolerant to isobutanol so that the yieldis not limited by butanol toxicity. Microbes that are metabolicallyactive at high titer levels of isobutanol are not well known in the art.Although butanol-tolerant mutants have been isolated from solventogenicClostridia, little information is available concerning the butanoltolerance of other potentially useful bacterial strains. Most of thestudies on the comparison of alcohol tolerance in bacteria suggest thatbutanol is more toxic than ethanol (de Cavalho, et al., Microsc. Res.Tech. 64, 215-22, 2004) and (Kabelitz, et al., FEMS Microbiol. Lett.220, 223-227, 2003, Tomas, et al. J. Bacteriol. 186, 2006-2018, 2004)report that the yield of 1-butanol during fermentation in Clostridiumacetobutylicum may be limited by 1-butanol toxicity. The primary effectof 1-butanol on Clostridium acetobutylicum is disruption of membranefunctions (Hermann et al., Appl. Environ. Microbiol. 50, 1238-1243,1985).

The microbial hosts selected for the production of isobutanol should betolerant to isobutanol and should be able to convert carbohydrates toisobutanol. The criteria for selection of suitable microbial hostsinclude the following: intrinsic tolerance to isobutanol, high rate ofglucose utilization, availability of genetic tools for genemanipulation, and the ability to generate stable chromosomalalterations.

Suitable host strains with a tolerance for isobutanol may be identifiedby screening based on the intrinsic tolerance of the strain. Theintrinsic tolerance of microbes to isobutanol may be measured bydetermining the concentration of isobutanol that is responsible for 50%inhibition of the growth rate (IC₅₀) when grown in a minimal medium. TheIC₅₀ values may be determined using methods known in the art. Forexample, the microbes of interest may be grown in the presence ofvarious amounts of isobutanol and the growth rate monitored by measuringthe optical density at 600 nanometers. The doubling time may becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of isobutanol thatproduces 50% inhibition of growth may be determined from a graph of thepercent inhibition of growth versus the isobutanol concentration.Preferably, the host strain should have an IC₅₀ for isobutanol ofgreater than about 0.5%.

The microbial host for isobutanol production should also utilize glucoseat a high rate. Most microbes are capable of utilizing carbohydrates.However, certain environmental microbes cannot utilize carbohydrates tohigh efficiency, and therefore would not be suitable hosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology may be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistance markers are available. The cloning vectors are tailoredto the host organisms based on the nature of antibiotic resistancemarkers that can function in that host.

The microbial host also has to be manipulated in order to inactivatecompeting pathways for carbon flow by deleting various genes. Thisrequires the availability of either transposons to direct inactivationor chromosomal integration vectors. Additionally, the production hostshould be amenable to chemical mutagenesis so that mutations to improveintrinsic isobutanol tolerance may be obtained.

Based on the criteria described above, suitable microbial hosts for theproduction of isobutanol include, but are not limited to, members of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferredhosts include: Escherichia coli, Alcaligenes eutrophus, Bacilluslichenifonnis, Paenibacillus macerans, Rhodococcus erythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis andSaccharomyces cerevisiae.

Construction of Production Host

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a fermentable carbonsubstrate to isobutanol may be constructed using techniques well knownin the art. In the present invention, genes encoding the enzymes of oneof the isobutanol biosynthetic pathways of the invention, for example,acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxyacid dehydratase, branched-chain α-keto acid decarboxylase, andbranched-chain alcohol dehydrogenase, may be isolated from varioussources, as described above.

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer-directedamplification methods such as polymerase chain reaction (U.S. Pat. No.4,683,202) to obtain amounts of DNA suitable for transformation usingappropriate vectors. Tools for codon optimization for expression in aheterologous host are readily available. Some tools for codonoptimization are available based on the GC content of the host organism.

Once the relevant pathway genes are identified and isolated they may betransformed into suitable expression hosts by means well known in theart. Vectors or cassettes useful for the transformation of a variety ofhost cells are common and commercially available from companies such asEPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.),Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly,Mass.). Typically the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. Both controlregions may be derived from genes homologous to the transformed hostcell, although it is to be understood that such control regions may alsobe derived from genes that are not native to the specific species chosenas a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements is suitable forthe present invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli, Alcaligenes, andPseudomonas) as well as the amy, apr, npr promoters and various phagepromoters useful for expression in Bacillus subtilis, Bacilluslicheniformis, and Paenibacillus macerans.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors-pRK437, pRK442,and pRK442(H) are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid 50, 74-79, 2003). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for the heterologous gene expression inGram-negative bacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to effect gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol. 174, 5633-5638, 1992). Additionally, invitro transposomes are available to create random mutations in a varietyof genomes from commercial sources such as EPICENTRE®.

The expression of an isobutanol biosynthetic pathway in variouspreferred microbial hosts is described in more detail below.

Expression of an Isobutanol Biosynthetic Pathway in E. Coli

Vectors or cassettes useful for the transformation of E. coli are commonand commercially available from the companies listed above. For example,the genes of an isobutanol biosynthetic pathway may be isolated fromvarious sources, cloned into a modified pUC19 vector and transformedinto E. coli NM522.

Expression of an Isobutanol Biosynthetic Pathway in RhodococcusErythropolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to, pRhBR17and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol. 62, 61-68,2003). Additionally, a series of promoters are available forheterologous gene expression in R. erythropolis (Nakashima et al., Appl.Environ. Microbiol. 70, 5557-5568, 2004 and Tao et al., Appl. Microbiol.Biotechnol. 68, 346-354, 2005). Targeted gene disruption of chromosomalgenes in R. erythropolis may be created using the method described byTao et al., supra, and Brans et al. (Appl. Environ. Microbiol. 66,2029-2036, 2000).

The heterologous genes required for the production of isobutanol, asdescribed above, may be cloned initially in pDA71 or pRhBR71 andtransformed into E. coli. The vectors may then be transformed into R.erythropolis by electroporation, as described by Kostichka et al.,supra. The recombinants may be grown in synthetic medium containingglucose and the production of isobutanol can be followed using methodsknown in the art.

Expression of an Isobutanol Biosynthetic Pathway in B. Subtilis

Methods for gene expression and creation of mutations in B. subtilis arealso well known in the art. For example, the genes of an isobutanolbiosynthetic pathway may be isolated from various sources, cloned into amodified pUC19 vector and transformed into Bacillus subtilis BE1010.Additionally, the five genes of an isobutanol biosynthetic pathway canbe split into two operons for expression. The three genes of the pathway(bubB, ilvD, and kivD) can be integrated into the chromosome of Bacillussubtilis BE1010 (Payne, et al., J. Bacteriol. 173, 2278-2282, 1991). Theremaining two genes (ilvC and bdhB) can be cloned into an expressionvector and transformed into the Bacillus strain carrying the integratedisobutanol genes

Expression of an Isobutanol Biosynthetic Pathway in B. Licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilismay be used to transform B. licheniformis by either protoplasttransformation or electroporation. The genes required for the productionof isobutanol may be cloned in plasmids pBE20 or pBE60 derivatives(Nagarajan et al., Gene 114,121-126, 1992). Methods to transform B.licheniformis are known in the art (Fleming et al. Appl. Environ.Microbiol., 61, 3775-3780, 1995). The plasmids constructed forexpression in B. subtilis may be transformed into B. licheniformis toproduce a recombinant microbial host that produces isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in PaenibacillusMacerans

Plasmids may be constructed as described above for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microbial host that producesisobutanol.

Expression of the Isobutanol Biosynthetic Pathway in Alcaligenes(Ralstonia) Eutrophus

Methods for gene expression and creation of mutations in Alcaligeneseutrophus are known in the art (Taghavi et al., Appl. Environ.Microbiol., 60, 3585-3591, 1994). The genes for an isobutanolbiosynthetic pathway may be cloned in any of the broad host rangevectors described above, and electroporated to generate recombinantsthat produce isobutanol. The poly(hydroxybutyrate) pathway inAlcaligenes has been described in detail, a variety of genetictechniques to modify the Alcaligenes eutrophus genome is known, andthose tools can be applied for engineering an isobutanol biosyntheticpathway.

Expression of an isobutanol biosynthetic pathway in Pseudomonas putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference). The butanol pathway genes may beinserted into pPCU18 and this ligated DNA may be electroporated intoelectrocompetent Pseudomonas putida DOT-T1C5aAR1 cells to generaterecombinants that produce isobutanol.

Expression of an Isobutanol Biosynthetic Pathway in SaccharomycesCerevisiae

Methods for gene expression in Saccharomyces cerevisiae are known in theart (e.g., Methods in Enzymology, Volume 194, Guide to Yeast Geneticsand Molecular and Cell Biology, Part A, 2004, Christine Guthrie andGerald R. Fink, eds., Elsevier Academic Press, San Diego, Calif.).Expression of genes in yeast typically requires a promoter, followed bythe gene of interest, and a transcriptional terminator. A number ofyeast promoters can be used in constructing expression cassettes forgenes encoding an isobutanol biosynthetic pathway, including, but notlimited to constitutive promoters FBA, GPD, ADH1, and GPM, and theinducible promoters GAL1, GAL10, and CUP1. Suitable transcriptionalterminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t,GAL1t, CYC1, and ADH1. For example, suitable promoters, transcriptionalterminators, and the genes of an isobutanol biosynthetic pathway may becloned into E. coli-yeast shuttle vectors.

Expression of an Isobutanol Biosynthetic Pathway in LactobacillusPlantarum

The Lactobacillus genus belongs to the Lactobacillus family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus may be used for lactobacillus. Non-limiting examples ofsuitable vectors include pAMβ1 and derivatives thereof (Renault et al.,Gene 183,175-182, 1996); and (O'Sullivan et al., Gene 137, 227-231,1993); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl.Environ. Microbiol. 62, 1481-1486, 1996); pMG1, a conjugative plasmid(Tanimoto et al., J. Bacteriol. 184, 5800-5804, 2002); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol. 63, 4581-4584, 1997);pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67, 1262-1267, 2001);and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38, 1899-1903,1994). Several plasmids from Lactobacillus plantarum have also beenreported (van Kranenburg R, et al. Appl. Environ. Microbiol. 71,1223-1230, 2005).

Expression of an Isobutanol Biosynthetic Pathway in Various Enterococcusspecies (E. faecium, E. gallinarium, and E. faecalis)

The Enterococcus genus belongs to the Lactobacillus family and manyplasmids and vectors used in the transformation of Lactobacilli, Bacilliand Streptococci species may be used for Enterococcus species.Non-limiting examples of suitable vectors include pAMβ1 and derivativesthereof (Renault et al., Gene 183,175-182, 1996); and (O'Sullivan etal., Gene 137, 227-231, 1993); pMBB1 and pHW800, a derivative of pMBB1(Wyckoff et al. Appl. Environ. Microbiol. 62, 1481-1486, 1996); pMG1, aconjugative plasmid (Tanimoto et al., J. Bacteriol. 184, 5800-5804,2002); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63,4581-4584, 1997); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67,1262-1267, 2001); and pAT392 (Arthur et al., Antimicrob. AgentsChemother. 38, 1899-1903, 1994). Expression vectors for E. faecalisusing the nisA gene from Lactococcus may also be used (Eichenbaum etal., Appl. Environ. Microbiol. 64, 2763-2769, 1998). Additionally,vectors for gene replacement in the E. faecium chromosome may be used(Nallaapareddy et al., Appl. Environ. Microbiol. 72, 334-345, 2006).

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. (eds):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153, 485-489, 1990). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forisobutanol production.

Culture Conditions

Typically cells are grown at a temperature in the range of about 25° C.to about 40° C. in an appropriate medium. Suitable growth media in thepresent invention are common commercially prepared media such as LuriaBertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM)broth. Other defined or synthetic growth media may also be used, and theappropriate medium for growth of the particular microorganism will beknown by one skilled in the art of microbiology or fermentation science.The use of agents known to modulate catabolite repression directly orindirectly, e.g., cyclic adenosine 2′,3′-monophosphate (cAMP), may alsobe incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred for the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classicalbatch fermentation is a closed system where the composition of themedium is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the medium is inoculated with the desired organismor organisms, and fermentation is permitted to occur without addinganything to the system. Typically, however, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund(Appl. Biochem. Biotechnol., 36, 227, 1992), herein incorporated byreference.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for isobutanol production.

Methods for Isobutanol Isolation from the Fermentation Medium

The biologically produced isobutanol may be isolated from thefermentation medium using methods known in the art forAcetone-butanol-ethanol (ABE) fermentations (see for example, Durre,Appl. Microbiol. Biotechnol. 49, 639-648, 1998), and (Groot et al.,Process. Biochem. 27, 61-75, 1992 and references therein). For example,solids may be removed from the fermentation medium by centrifugation,filtration, decantation and isobutanol may be isolated from thefermentation medium using methods such as distillation, azeotropicdistillation, liquid-liquid extraction, adsorption, gas stripping,membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984,and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Materialsand Methods suitable for the maintenance and growth of bacterialcultures are also well known in the art. Techniques suitable for use inthe following Examples may be found in Manual of Methods for GeneralBacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow,Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips,eds., American Society for Microbiology, Washington, D.C., 1994, or byThomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology,Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. Allreagents, restriction enzymes and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), LifeTechnologies (Rockville, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

The meaning of abbreviations used is as follows: “Å” means Angstrom,“min” means minute(s), “h” means hour(s), “μl” means microliter(s),“ng/μl” means nano gram per microliter, “pmol/μl” means pico mole permicroliter, “ml” means milliliter(s), “L” means liter(s), “g/L” meangram per liter, “ng” means nano gram, “sec” means second(s), “ml/min”means milliliter per minute(s), “w/v” means weight per volume, “v/v”means volume per volume, “nm” means nanometer(s), “mm” meansmillimeter(s), “cm” means centimeter(s) “mM” means millimolar, “M” meansmolar, “mmol” means millimole(s), “μmole” means micromole(s), “g” meansgram(s), “μg” means microgram(s), “mg” means milligram(s), “g” means thegravitation constant, “rpm” means revolutions per minute, “HPLC” meanshigh performance liquid chromatography, “MS” means mass spectrometry,“HPLC/MS” means high performance liquid chromatography/massspectrometry, “EDTA” means ethylendiamine-tetraacetic acid, “dNTP” meansdeoxynucleotide triphosphate.

The oligonucleotide primers used in the following Examples have beendescribed herein (see Table 1)

High Throughput Screening Assay of Gene Libraries

High throughput screening of the gene libraries of mutant KARI enzymeswas performed as described herein: 10× freezing medium containing 554.4g/L glycerol, 68 mM of (NH₄)₂SO₄, 4 mM MgSO₄, 17 mM sodium citrate, 132mM KH₂PO₄, 36 mM K₂HPO₄ was prepared with molecular pure water andfilter-sterilized. Freezing medium was prepared by diluting the 10×freezing medium with the LB medium. An aliquot (200 μl) of the freezingmedium was used for each well of the 96-well archive plates (cat #3370,Corning Inc. Corning, N.Y.).

Clones from the LB agar plates were selected and inoculated into the96-well archive plates containing the freezing medium and grownovernight at 37° C. without shaking. The archive plates were then storedat −80° C. E. coli strain Bw25113 transformed with pBAD-HisB(Invitrogen) was always used as the negative control. For libraries C,E, F and G, mutant T52D of (PF5-ilvC) was used as the positive control.The mutant T52D was a mutant of PF5-ilvC in which the threonine atposition 52 was changed to aspartic acid. For library H, mutant C3B11(R47F/S50A/T52D/v53W of PF5-ilvC) was used as the positive control.

Clones from archive plates were inoculated into the 96-deep well plates.Each well contained 3.0 μl of cells from thawed archive plates, 300 μlof the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v)arabinose as the inducer. Cells were the grown overnight at 37° C. with80% humidity while shaking (900 rpm), harvested by centrifugation (4000rpm, 5 min at 25° C.). (Eppendorf centrifuge, Brinkmann Instruments,Inc. Westbury, N.Y.) and the cell pellet was stored at −20° C. for lateranalysis.

The assay substrate, (R,S)-acetolactate, was synthesized as described byAulabaugh and Schloss (Aulabaugh and Schloss, Biochemistry, 29,2824-2830, 1990): 1.0 g of 2-acetoxy-2-methyl-3-oxobutyric acid ethylester (Aldrich, Milwaukee, Wis.) was mixed with 10 ml NaOH (1.0 M) andstirred at room temperature. When the solution's pH became neutral,additional NaOH was slowly added until pH˜8.0 was maintained. All otherchemicals used in the assay were purchased from Sigma.

The enzymatic conversion of acetolactate to α-dihydroxy-isovalerate byKARI was followed by measuring the disappearance of the cofactor, NADPHor NADH, from the reaction at 340 nm using a plate reader (MolecularDevice, Sunnyvale, Calif.). The activity was calculated using the molarextinction coefficient of 6220 M cm⁻¹ for either NADPH or NADH. Thestock solutions used were: K₂HPO₄ (0.2 M); KH₂PO₄ (0.2 M); EDTA (0.5 M);MgCl₂ (1.0 M); NADPH (2.0 mM); NADH (2.0 mM) and acetolactate (45 mM).The 100 ml reaction buffer mix stock containing: 4.8 ml K₂HPO₄, 0.2 mlKH₂PO₄, 4.0 ml MgCl₂, 0.1 ml EDTA and 90.9 ml water was prepared.

Frozen cell pellet in deep-well plates and BugBuster were warmed up atroom temperature for 30 min at the same time. Each well of 96-well assayplates was filled with 120 μl of the reaction buffer and 20 μl of NADH(2.0 mM), 150 μl of BugBuster was added to each well after 30 minwarm-up and cells were suspended using Genmate (Tecan Systems Inc. SanJose, Calif.) by pipetting the cell suspension up and down (×5). Theplates were incubated at room temperature for 20 min and then heated at60° C. for 10 min. The cell debris and protein precipitates were removedby centrifugation at 4,000 rpm for 5 min at 25° C. An aliquot (50 μl) ofthe supernatant was transferred into each well of 96-well assay plates,the solution was mixed and the bubbles were removed by centrifugation at4000 rpm at 25° C. for 1 min. Absorbance at 340 nm was recorded asbackground, 20 μl of acetolactate (4.5 mM, diluted with the reactionbuffer) was added to each well and mixed with shaking by the platereader. Absorbance at 340 nm was recoded at 0, and 60 minutes aftersubstrate addition. The difference in absorbance (before and aftersubstrate addition) was used to determine the activity of the mutants.Mutants with higher KARI activity compared to the wild type wereselected for re-screening.

About 5,000 clones were screened for library C and 360 top performerswere selected for re-screen. About 92 clones were screened for library Eand 16 top performers were selected for re-screening. About 92 cloneswere screened for library F and 8 top performers were selected forre-screening. About 92 clones were screened for library G and 20 topperformers were selected for re-screening. About 8,000 clones werescreened for library H and 62 top performers were selected forre-screening.

For library C, about 360 top performers were re-screened using the sameprocedure as for the general screening. Among them, 45 top performerswere further selected for re-screening as described below.

Secondary Assay of Active Mutants

Cells containing pBad-ilvC and its mutants identified by high throughputscreening were grown overnight, at 37° C., in 3.0 ml of the LB mediumcontaining 100 μg/ml ampicillin and 0.02% (w/v) arabinose as the inducerwhile shaking at 250 rpm. The cells were then harvested bycentrifugation at 18,000×g for 1 min at room temperature (Sigmamicro-centrifuge model 1-15, Laurel, Md.). The cell pellets werere-suspended in 300 μl of BugBuster Master Mix (EMD Chemicals). Thereaction mixture was first incubated at room temperature for 20 min andthen heated at 60° C. for 10 min. The cell debris and proteinprecipitate were removed by centrifugation at 18,000×g for 5 min at roomtemperature.

The reaction buffer (120 μl) prepared as described above was mixed witheither NADH or NADPH (20 μl) stock and cell extract (20 μl) in each wellof a 96-well assay plate. The absorbance at 340 nm at 25° C. wasrecorded as background. Then 20 μl of acetolactate (4.5 mM, diluted withreaction buffer) was added each well and mixed with shaking by the platereader. The absorbance at 340 nm at 0 min, 2 min and 5 min after addingacetolactate was recorded. The absorbance difference before and afteradding substrate was used to determine the activity of the mutants. Themutants with high activity were selected for sequencing.

Five top performers from “Library C” were identified and sequenced (FIG.4). The best performer was mutant R47F/S50A/T52D/V53W, which completelyreversed the nucleotide specificity. The best performers from “LibrariesE, F and G” were R47P, S50D and T52D respectively (FIG. 5). For “LibraryH”, 5 top performers were identified and sequenced (FIG. 6) and the bestperformer was R47P/S50G/T52D, which also completely reversed thenucleotide specificity. Enzymes containing activities higher than thebackground were considered positive.

Kari Enzyme Assay

KARI enzyme activity can be routinely measured by NADH or NADPHoxidation as described above, however to measure formation of the2,3-dihydroxyisovalerate product directly, analysis of the reaction wasperformed using LC/MS.

Protein concentration of crude cell extract from Bugbuster lysed cells(as described above) was measured using the BioRad protein assay reagent(BioRad Laboratories, Inc., Hercules, Calif. 94547). A total of 0.5micrograms of crude extract protein was added to a reaction bufferconsisting of 100 mM HEPES-KOH, pH 7.5, 10 mM MgCl₂, 1 mMglucose-6-phosphate (Sigma-Aldrich), 0.2 Units of Leuconostocmesenteroides glucose-6-phosphate dehydrogenase (Sigma-Aldrich), andvarious concentrations of NADH or NADPH, to a volume of 96 μL. Thereaction was initiated by the addition of 4 μL of acetolactate to afinal concentration of 4 mM and a final volume of 100 μL. After timedincubations at 30° C., typically between 2 and 15 min, the reaction wasquenched by the addition of 10 μL of 0.5 M EDTA pH 8.0 (LifeTechnologies, Grand Island, N.Y. 14072). To measure the K_(M) of NADH,the concentrations used were 0.03, 0.1, 0.3, 1, 3, and 10 mM.

To analyze for 2,3-dihydroxyisovalerate, the sample was diluted 10× withwater, and 8.0 μl was injected into a Waters Acquity HPLC equipped withWaters SQD mass spectrometer (Waters Corporation, Milford, Mass.). Thechromatography conditions were: flow rate (0.5 ml/min), on a WatersAcquity HSS T3 column (2.1 mm diameter, 100 mm length). Buffer Aconsisted of 0.1% (v/v) in water, Buffer B was 0.1% formic acid inacetonitrile. The sample was analyzed using 1% buffer B (in buffer A)for 1 min, followed by a linear gradient from 1% buffer B at 1 min to75% buffer B at 1.5 min. The reaction product,2,3-dihydroxyiso-valerate, was detected by ionization at m/z=133, usingthe electrospay ionization devise at −30 V cone voltage. The amount ofproduct 2,3-dihydroxyisovalerate was calculated by comparison to anauthentic standard.

To calculate the K_(M) for NADH, the rate data for DHIV formation wasplotted in Kaleidagraph (Synergy Software, Reading, Pa.) and fitted tothe single substrate Michaelis-Menton equation, assuming saturatingacetolactate concentration.

Example 1 Construction of Site-Saturation Gene Libraries

Seven gene libraries were constructed (Table 2) using two steps: 1)synthesis of MegaPrimers using commercially synthesizedoligonucleotidies described in Table 1; and 2) construction of mutatedgenes using the MegaPrimers obtained in step 1. These primers wereprepared using high fidelity pfu-ultra polymerase (Stratagene, La Jolla,Calif.) for one pair of primer containing one forward and one reverseprimer. The templates for libraries C, E, F, G and H were the wild typeof PF5_ilvc. The DNA templates for library N were those mutants havingdetectable NADH activity from library C while those for library 0 werethose mutants having detectable NADH activity from library H. A 50 μlreaction mixture contained: 5.0 μl of 10× reaction buffer supplied withthe pfu-ultra polymerase (Stratagene), 1.0 μl of 50 ng/μl template, 1.0μl each of 10 pmol/μl forward and reverse primers, 1.0 μl of 40 mM dNTPmix (Promega, Madison, Wis.), 1.0 μl pfu-ultra DNA polymerase(Stratagene) and 39 μl water. The mixture was placed in a thin well 200μl tube for the PCR reaction in a Mastercycler gradient equipment(Brinkmann Instruments, Inc. Westbury, N.Y.). The following conditionswere used for the PCR reaction: The starting temperature was 95° C. for30 sec followed by 30 heating/cooling cycles. Each cycle consisted of95° C. for 30 sec, 54° C. for 1 min, and 70° C. for 2 min. At thecompletion of the temperature cycling, the samples were kept at 70° C.for 4 min more, and then held awaiting sample recovery at 4° C. The PCRproduct was cleaned up using a DNA cleaning kit (Cat#D4003, ZymoResearch, Orange, Calif.) as recommended by the manufacturer.

TABLE 2 GENE LIBRARIES Library Targeted position(s) name Templates ofPf5_ilvC Primers used C PF5_ilvc 47, 50, 52 and 53 SEQ ID No: 1 and 2 EPF5_ilvc 47 SEQ ID No: 1 and 3 F PF5_ilvc 50 SEQ ID No: 1 and 4 GPF5_ilvc 52 SEQ ID No: 1 and 5 H PF5_ilvc 47, 50, and 52 SEQ ID No: 1and 6 N Good mutants 53 SEQ ID NO: 20 and 21 from library C O Goodmutants 53 SEQ ID NO: 20 and 21 from library H

The MegaPrimers were then used to generate gene libraries using theQuickChange II XL site directed mutagenesis kit (Catalog #200524,Stratagene, La Jolla Calif.). A 50 μl reaction mixture contained: 5.0 μlof 10× reaction buffer, 1.0 μl of 50 ng/μl template, 42 μl Megaprimer,1.0 μl of 40 mM dNTP mix, 1.0 μl pfu-ultra DNA polymerase. Except forthe MegaPrimer and the templates, all reagents used here were suppliedwith the kit indicated above. This reaction mixture was placed in a thinwell 200 μl-capacity PCR tube and the following reactions were used forthe PCR: The starting temperature was 95° C. for 30 sec followed by 25heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55°C. for 1 min, and 68° C. for 6 min. At the completion of the temperaturecycling, the samples were kept at 68° C. for 8 min more, and then heldat 4° C. for later processing. Dpn I restriction enzyme (1.0 μl)(supplied with the kit above) was directly added to the finishedreaction mixture, enzyme digestion was performed at 37° C. for 1 hr andthe PCR product was cleaned up using a DNA cleaning kit (Zymo Research).The cleaned PCR product (10 μl) contained mutated genes for a genelibrary.

The cleaned PCR product was transformed into an electro-competent strainof E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-RadLaboratories Inc., Hercules, Calif.). The transformed clones werestreaked on agar plates containing the Lauria Broth medium and 100 μg/mlampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at37° C. overnight. Dozens of clones were randomly chosen for DNAsequencing to confirm the quality of the library.

TABLE 3 List of some mutants having NADH activities identified fromsaturation libraries Mutant Position 47 Position 50 Position 52 Position53 SD2 R47Y S50A T52H V53W SB1 R47Y S50A T52G V53W SE1 R47A S50W T52GV53W SH2 R47N S50W T52N V53W SB2 R47I T52G V53W SG1 R47Y T52G V53W SB3R47G S50W T52G V53W SE2 R47P S50E T52A V53W SD3 R47L S50W T52G V53W C2A6R47I S50G T52D V53W C3E11 R47A S50M T52D V53W C3A7 R47Y S50A T52D V53WC3B11 R47F S50A T52D V53W C4A5 R47Y S50A T52S V53W C3B12 R47I T52D V53WC4H7 R47I T52S V53W C1D3 R47G S50M T52D V53W C4D12 R47C S50W T52G V53WC1G7 R47P S50G T52D V53W C2F6 R47P S50V T52D V53W C1C4 R47P S50E T52SV53W 6924F9 R47P S50G T52D 6881E11 R47P S50N T52C 6868F10 R47P T52S6883G10 R47P S50D T52S 6939G4 R47P S50C T52D 11463D8 R47P S50F T52D9667A11 R47N S50N T52D V53A 9675C8 R47Y S50A T52D V53A 9650E5 R47N S50WT52G V53H 9875B9 R47N S50N T52D V53W 9862B9 R47D S50W T52G V53W 9728G11R47N S50W T52G V53W 11461D8 R47F S50A T52D V53A 11461A2 R47P S50F T52DV53I

Example 2 Construction of Error Prone PCR Libraries

Several rounds of error prone PCR (epPCR) libraries were created usingthe GeneMorph II kit (Stratagene) as recommended by the manufacturer.All the epPCR libraries target the N-terminal of PF5—KARI. The forwardprimer (SED ID No: 20) and the reverse primer (SED ID No: 22) were usedfor all ePCR libraries.

The DNA templates for each epPCR library were mutants having relativelygood NADH activities from the previous library. For example: the DNAtemplates for the n^(th) epPCR library were mutants having good NADHactivities from the (n-1)th epPCR library. The templates of the firstepPCR library were mutants having relatively good NADH activities fromlibraries N and O. The mutations rate of library made by this kit wascontrolled by the amount of template added in the reaction mixture andthe number of amplification cycles. Typically, 1.0 ng of each DNAtemplate was used in 100 μl of reaction mixture. The number ofamplification cycles was 70. The following conditions were used for thePCR reaction: The starting temperature was 95° C. for 30 sec followed by70 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec,55° C. for 30 min, and 70° C. for 2 min. After the first 35heating/cooling cycles finished, more dNTP and Mutazyme II DNApolymerase were added. The PCR product was cleaned up using a DNAcleaning kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommendedby the manufacturer. The cleaned PCR product was treated as megaprimerand introduced into the vector using the Quickchange kit as described inExample 1. Table 4 below lists the KARI mutants obtained and thesignificant improvement observed in their binding NADH. The K_(m) wasreduced from 1100 μM for mutant C3B11 to 50 μM for mutant 12957G9.

TABLE 4 List of some mutants with their measured K_(m) values NADHMutant Mutation Locations K_(m) (μM) C3B11 R47F/S50A/T52D/V53W 1100 SB3R47G/S50W/T52G/V53W 500 11518B4 R47N/S50N/T52D/V53A/A156V 141 11281G2R47N/S50N/T52D/V53A/A156V/L165M 130 12985F6R47Y/S50A/T52D/V53A/L61F/A156V 100 13002D8R47Y/S50A/T52D/V53A/L61F/A156V/G170A 68 12957G9Y24F/R47Y/S50A/T52D/V53A/L61F/G170A 50 12978D9R47Y/S50A/T52D/V53A/L61F/Q115L/A156V 114

Example 3 Thermostability of PF5-ilvC and its Mutants

Cells containing mutated pBad-ilvC were grown overnight at 37° C. in 25ml of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v)arabinose inducer while shaking at 250 rpm. The cells were thenharvested by centrifugation at 18,000×g for 1 min at room temperatureand the cell pellets were re-suspended in 300 μl of BugBuster Master Mix(EMD Chemicals). The reaction mixture was first incubated at roomtemperature for 20 min and aliquots of this cell mixture (e.g. 50 μl)were incubated at different temperatures (from room temperature to 75°C.) for 10 min. The precipitate was removed by centrifugation at18,000×g for 5 min at room temperature. The remaining activity of thesupernatant was analyzed as described above. As shown in FIG. 7,pBad-ilvC was very stable with T₅₀ equal to 68° C. (T₅₀ is thetemperature, at which 50% of protein lost its activity after 10 minincubation).

The thermostability of PF5-ilvC allowed destruction of most of the othernon-KARI NADH oxidation activity within these cells, reducing the NADHbackground consumption and thus facilitating the KARI activity assays.This heat treatment protocol was used in all screening and re-screeningassays. The mutants thus obtained were all thermostable which allowedeasier selection of the desirable mutants.

Example 4 Stoichiometric Production of 2,3-Dihydroxyisovalerate by KariDuring Consumption of Cofactors NADH or NADPH

Screening and routine assays of KARI activity rely on the 340 nmabsorption decrease associated with oxidation of the pyridinenucleotides NADPH or NADH. To insure that this metric was coupled toformation of the other reaction product, oxidation of pyridinenucleotide and formation of 2,3-dihydroxyisovalerate were measured inthe same samples.

The oxidation of NADH or NADPH was measured at 340 nm in a 1 cm pathlength cuvette on a Agilent model 8453 spectrophotometer (AgilentTechnologies, Wilmington Del.). Crude cell extract (0.1 ml) prepared asdescribed above containing either wild type PF5 KARI or the C3B11mutant, was added to 0.9 ml of K-phosphate buffer (10 mM, pH 7.6),containing 10 mM MgCl₂, and 0.2 mM of either NADPH or NADH. The reactionwas initiated by the addition of acetolactate to a final concentrationof 0.4 mM. After 10-20% decrease in the absorption (about 5 min), 50 μlof the reaction mixture was rapidly withdrawn and added to a 1.5 mlEppendorf tube containing 10 μl 0.5 mM EDTA to stop the reaction and theactual absorption decrease for each sample was accurately recorded.Production of 2,3-dihydroxyisovalerate was measured and quantitated byLC/MS as described above.

The coupling ratio is defined by the ratio between the amount of2,3-dihydroxyisovalerate (DHIV) produced and the amount of either NADHor NADPH consumed during the experiment. The coupling ratio for the wildtype enzyme (PF5-ilvC), using NADPH, was 0.98 DHIV/NADPH, while that forthe mutant (C3B11), using NADH, was on average around 1.10 DH IV/NADPH.

Example 5 Identification of Amino Acids Involved in Cofactor Binding inKari for Cofactor Specificity Switching Using Bioinformatic Tools

To discover if naturally existing KARI sequences could provide clues foramino acid positions that should be targeted for mutagenesis, multiplesequence alignment (MSA) using PF5_KARI, its close homolog PAO1_KARI andthree KARI sequences with measurable NADH activity, i.e., B. CereusilvC1 and ilvC2 and spinach KARI were performed (FIG. 8). Based on themultiple sequence alignment, positions 33, 43, 59, 61, 71, 80, 101, and119 were chosen for saturation mutagenesis. Saturation mutagenesis onall of these positions was performed simultaneously using theQuickChange II XL site directed mutagenesis kit (Catalog #200524,Stratagene, La Jolla Calif.) with the manufacturer's suggested protocol.Starting material for this mutagenesis was a mixed template consistingof the mutants already identified in Table 4 and Example 2. The primersused are listed in Table 5. The library of mutants thus obtained werenamed library Z″. Mutants with good NADH activity from this library wereidentified using high throughput screening and their KARI activity andthe K_(m) for NADH were measured as described above. These mutants(listed in Table 6) possess much lower K_(m)s for NADH compared to theparent templates (Table 4). A megaprimer, using primers (SEQ ID Nos. 20and 58), was created and mutations at positions 156 and 170 wereeliminated. Further screening of this set of mutants identified mutant3361 G8 (SEQ ID NO: 67)(Table 7). The hits from library Z were furthersubjected to saturation mutagenesis at position 53 using primers (SEQ IDNos. 20 and 21), and subsequent screening identified the remainingmutants in Table 7. As shown in Table 7 the new mutants possessed muchlower K_(m) for NADH (e.g., 4.0 to 5.5 μM) compared to mutants listed inTable 6 (e.g., 14-40 M).

TABLE 5 Primers for Example 5 Targeted position(s) of Pf5_ilvC Primers33 pBAD-405-C33_090808f: GCTCAAGCANNKAACCTGAAGG (SEQ ID NO: 49)pBAD-427-C33_090808r: CCTTCAGGTTKNNTGCTTGAGC (SEQ ID NO: 50) 43pBAD-435-T43_090808f: GTAGACGTGNNKGTTGGCCTG (SEQ ID NO: 51)pBAD-456-T43_090808r: CAGGCCAACKNNCACGTCTAC (SEQ ID NO: 52) 59 and 61pBAD-484-H59L61_090808f: CTGAAGCCNNKGGCNNKAAAGTGAC (SEQ ID NO: 53)pBAD-509-H59L61_090808r: GTCACTTTKNNGCCKNNGGCTTCAG (SEQ ID NO: 54) 71pBAD-519-A71_090808f: GCAGCCGTTNNKGGTGCCGACT (SEQ ID NO: 55)pBAD-541-A71_090808r: AGTCGGCACCKNNAACGGCTGC (SEQ ID NO: 56) 80pBAD-545-T80_090808f: CATGATCCTGNNKCCGGACGAG (SEQ ID NO: 57)pBAD-567-T80_090808r: CTCGTCCGGKNNCAGGATCATG (SEQ ID NO: 58) 101pBAD-608-A101_090808f: CAAGAAGGGCNNKACTCTGGCCT (SEQ ID NO: 59)pBAD-631-A101_090808r: AGGCCAGAGTKNNGCCCTTCTTG (SEQ ID NO: 60) 119pBAD-663-R119_090808f: GTTGTGCCTNNKGCCGACCTCG (SEQ ID NO: 61)pBAD-685-R119_090808r: CGAGGTCGGCKNNAGGCACAAC (SEQ ID NO: 62)

TABLE 6 List of some mutants with their measured K_(m) values (themutated positions in those mutants were indentified by bioinformatictools) NADH Mutant Mutation Locations K_(m) (μM) ZB1Y24F/R47Y/S50A/T52D/V53A/L61F/A156V 40 (SEQ ID NO: 24) ZF3Y24F/C33L/R47Y/S50A/T52D/V53A/L61F 21 (SEQ ID NO: 25) ZF2Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V 17 (SEQ ID NO: 26) ZB3Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A 17 (SEQ ID NO: 27) Z4B8C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V 14 (SEQ ID NO: 28)

TABLE 7 Mutants further optimized for improved K_(m) (for NADH) NADHMutant Mutation Locations K_(m) (μM) 3361G8C33L/R47Y/S50A/T52D/V53A/L61F/T80I 5.5 (SEQ ID NO: 67) 2H10Y24F/C33L/R47Y/S50A/T52D/V53I/L61F/T80I/ 5.3 A156V (SEQ ID NO: 68) 1D2Y24F/R47Y/S50A/T52D/V53A/L61F/T80I/A156V 4.1 (SEQ ID NO: 69) 3F12Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T80I/ 4.0 A156V (SEQ ID NO: 70)

Further analyses using bioinformatic tools were therefore performed toexpand the mutational sites to other KARI sequences as described below.

Sequence Analysis

Members of the protein family of ketol-acid reducoisomorase (KARI) wereidentified through BlastP searches of publicly available databases usingamino acid sequence of Pseudomonas fluorescens PF5 KARI (SEQ ID NO:17)with the following search parameters: E value=10, word size=3,Matrix=Blosum62, and Gap opening=11 and gap extension=1, E value cutoffof 10⁻³. Identical sequences and sequences that were shorter than 260amino acids were removed. In addition, sequences that lack the typicalGxGXX(G/A) motif involved in the binding of NAD(P)H in the N-terminaldomain were also removed. These analyses resulted in a set of 692 KARIsequences.

A profile HMM was generated from the set of the experimentally verifiedClass I and Class II KARI enzymes from various sources as described inTable 8. Details on building, calibrating, and searching with thisprofile HMM are provided below. Any sequence that can be retrieved byHMM search using the profile HMM for KARI at E-value above 1E⁻³ isconsidered a member of the KARI family. Positions in a KARI sequencealigned to the following in the profile HMM nodes (defined below in thesection of profile HMM building) are claimed to be responsible for NADHutilization: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 (thenumbering is based on the sequences of Pseudomonas fluorescens PF5KARI).

Preparation of Profile HMM

A group of KARI sequences were expressed in E. coli and have beenverified to have KARI activity These KARIs are listed in Table 6. Theamino acid sequences of these experimentally verified functional KARIswere analyzed using the HMMER software package (The theory behindprofile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G.Mitchison, Biological sequence analysis: probabilistic models ofproteins and nucleic acids, Cambridge University Press, 1998; Krogh etal., 1994; J. Mol. Biol. 235:1501-1531), following the user guide whichis available from: HMMER (Janelia Farm Research Campus, Ashburn, Va.).The output of the HMMER software program is a profile Hidden MarkovModel (profile HMM) that characterizes the input sequences. As stated inthe user guide, profile HMMs are statistical descriptions of theconsensus of a multiple sequence alignment. They use position-specificscores for amino acids (or nucleotides) and position specific scores foropening and extending an insertion or deletion. Compared to otherprofile based methods, HMMs have a formal probabilistic basis. ProfileHMMs for a large number of protein families are publicly available inthe PFAM database (Janelia Farm Research Campus, Ashburn, Va.).

The profile HMM was built as follows:

Step 1. Build a Sequence Alignment

The 25 sequences for the functionally verified KARIs listed above werealigned using Clustal W (Thompson, J. D., Higgins, D. G., and Gibson T.J. (1994) Nuc. Acid Res. 22: 46734680) with default parameters. Thealignment is shown in FIG. 9.

TABLE 8 25 Experimentally verified KARI enzymes GI Number Accession SEQID NO: Organism 70732562 YP_262325.1 17 Pseudomonas fluorescens Pf-515897495 NP_342100.1 13 Sulfolobus solfataricus P2 18313972 NP_560639.114 Pyrobaculum aerophilum str. IM2 76801743 YP_326751.1 30 Natronomonaspharaonis DSM 2160 16079881 NP_390707.1 31 Bacillus subtilis subsp.subtilis str. 168 19552493 NP_600495.1 32 Corynebacterium glutamicumATCC 13032 6225553 O32414 33 Phaeospririlum molischianum 17546794NP_520196.1 15 Ralstonia solanacearum GMI1000 56552037 YP_162876.1 34Zymomonas mobilis subsp. mobilis ZM4 114319705 YP_741388.1 35Alkalilimnicola ehrlichei MLHE-1 57240359 ZP_00368308.1 36 Campylobacterlari RM2100 120553816 YP_958167.1 37 Marinobacter aquaeolei VT8 71065099YP_263826.1 38 Psychrobacter arcticus 273-4 83648555 YP_436990.1 39Hahella chejuensis KCTC 2396 74318007 YP_315747.1 40 Thiobacillusdenitrificans ATCC 25259 67159493 ZP_00420011.1 41 Azotobactervinelandii AvOP 66044103 YP_233944.1 42 Pseudomonas syringae pv.syringae B728a 28868203 NP_790822.1 43 Pseudomonas syringae pv. tomatostr. DC3000 26991362 NP_746787.1 44 Pseudomonas putida KT2440 104783656YP_610154.1 45 Pseudomonas entomophila L48 146306044 YP_001186509.1 46Pseudomonas mendocina ymp 15599888 NP_253382.1 16 Pseudomonas aeruginosaPAO1 42780593 NP_977840.1 47 Bacillus cereus ATCC 10987 42781005NP_978252.1 48 Bacillus cereus ATCC 10987 266346 Q01292 18 Spinaciaoleracea

Step 2. Build a Profile HMM

The hmmbuild program was run on the set of aligned sequences usingdefault parameters. hmmbuild reads the multiple sequence alignment file,builds a new profile HMM, and saves the profile HMM to file. Using thisprogram an un-calibrated profile was generated from the multiplesequence alignment for twenty-four experimentally verified KARIs asdescribed above.

The following information based on the HMMER software user guide givessome description of the way that the hmmbuild program prepares a profileHMM. A profile HMM is a linear state machine consisting of a series ofnodes, each of which corresponds roughly to a position (column) in themultiple sequence alignment from which it is built. If gaps are ignored,the correspondence is exact, i.e., the profile HMM has a node for eachcolumn in the alignment, and each node can exist in one state, a matchstate. The word “match” here implies that there is a position in themodel for every position in the sequence to be aligned to the model.Gaps are modeled using insertion (I) states and deletion (D) states. Allcolumns that contain more than a certain fraction x of gap characterswill be assigned as an insert column. By default, x is set to 0.5. Eachmatch state has an I and a D state associated with it. HMMER calls agroup of three states (M/D/I) at the same consensus position in thealignment a “node”.

A profile HMM has several types of probabilities associated with it. Onetype is the transition probability—the probability of transitioning fromone state to another. There are also emissions probabilities associatedwith each match state, based on the probability of a given residueexisting at that position in the alignment. For example, for a fairlywell-conserved column in an alignment, the emissions probability for themost common amino acid may be 0.81, while for each of the other 19 aminoacids it may be 0.01.

A profile HMM is completely described in a HMMER2 profile save file,which contains all the probabilities that are used to parameterize theHMM. The emission probabilities of a match state or an insert state arestored as log-odds ratio relative to a null model: log₂ (p_x)/(null_x).Where p_x is the probability of an amino acid residue, at a particularposition in the alignment, according to the profile HMM and null_x isthe probability according to the Null model. The Null model is a simpleone state probabilistic model with pre-calculated set of emissionprobabilities for each of the 20 amino acids derived from thedistribution of amino acids in the SWISSPROT release 24. Statetransition scores are also stored as log odds parameters and areproportional to log₂(t_x). Where t_x is the transition probability oftransiting from one state to another state.

Step 3. Calibrate the Profile HMM

The profile HMM was read using hmmcalibrate which scores a large numberof synthesized random sequences with the profile (the default number ofsynthetic sequences used is 5,000), fits an extreme value distribution(EVD) to the histogram of those scores, and re-saves the HMM file nowincluding the EVD parameters. These EVD parameters (μ and λ) are used tocalculate the E-values of bit scores when the profile is searchedagainst a protein sequence database. Hmmcalibrate writes two parametersinto the HMM file on a line labeled “EVD”: these parameters are the p(location) and λ(scale) parameters of an extreme value distribution(EVD) that best fits a histogram of scores calculated on randomlygenerated sequences of about the same length and residue composition asSWISS-PROT. This calibration was done once for the profile HMM.

The calibrated profile HMM for the set of KARI sequences is providedappended hereto as a profile HMM Excel chart (Table 9). In the mainmodel section starting from the HMM flag line, the model has three linesper node, for M nodes (where M is the number of match states, as givenby the LENG line). The first line reports the match emission log-oddsscores: the log-odds ratio of emitting each amino acid from that stateand from the Null model. The first number if the node number (1.M). Thenext K numbers for match emission scores, one per amino acid. Thehighest scoring amino acid is indicated in the parenthesis after thenode number. These log-odds scores can be converted back to HMMprobabilities using the null model probability. The last number on theline represents the alignment column index for this match state. Thesecond line reports the insert emission scores, and the third linereports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D;B→M; M→E.

Step 4. Test the Specificity and Sensitivity of the Built Profile HMMs

The Profile HMM was evaluated using hmmsearch, which reads a Profile HMMfrom hmmfile and searches a sequence file for significantly similarsequence matches. The sequence file searched contained 692 sequences(see above). During the search, the size of the database (Z parameter)was set to 1 billion. This size setting ensures that significantE-values against the current database will remain significant in theforeseeable future. The E-value cutoff was set at 10.

A hmmer search, using hmmsearch, with the profile HMM generated from thealignment of the twenty-five KARIs with experimentally verifiedfunction, matched all 692 sequences with an E value<10⁻³. This resultindicates that members of the KARI family share significant sequencesimilarity. A hmmer search with a cutoff of E value 10⁻³ was used toseparate KARIs from other proteins.

Step 5. Identify Positions that are Relevant for NAD(P)H Utilization.

Eleven positions have been identified in KARI of Pseudomonas fluorescensPf-5 that switches the cofactor from NADPH to NADH. Since the KARIsequences share significant sequence similarity (as described above), itcan be reasoned that the homologous positions in the alignment of KARIsequences should contribute to the same functional specificity. Theprofile HMM for KARI enzymes has been generated from the multiplesequence alignment which contains the sequence of Pseudomonasfluorescens Pf-5 KARI. The eleven positions in the profile HMMrepresenting the columns in the alignment which correspond to the elevencofactor switching positions in Pseudomonas fluorescens Pf-5 KARI areidentified as positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and170. The lines corresponding to these positions in the model file arehighlighted in yellow in Table 9.

For any query sequence, hmm search is used to search the profile HMM forKARI against the query sequence and the alignment of the query to theHMM is recorded in the output file. In the alignment section of theoutput, the top line is the HMM consensus. The amino acid shown for theconsensus is the highest probability amino acid at that positionaccording to the HMM (not necessarily the highest scoring amino acid).The center line shows letters for “exact” matches to the highestprobability residue in the HMM, or a “+” when the match has a positivescore. The third line shows the sequence itself. The positions in thequery sequence that are deemed as relevant for cofactor switching areidentified as those that are aligned to these eleven nodes in theprofile HMM as described above. An example of the alignment ofPseudomonas fluorescens Pf-5 KARI to the profile HMM of KARI is shown inFIG. 10 and the eleven positions that are responsible for cofactorswitching are shaded in grey.

TABLE 9 A C D E F G H I K HMM m->m m->i m->d i->m i->i d->m d->d b->mm->e L M N −650 * −1463  1(Q) −648 −1356 −136 −44 −1453 −1166 −219 −1455321 −1417 −911 −227 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −38 −5840 −6882 −894 −1115 −701 −1378 −650 *  2(M) −4231 −3929−5216 −5402 −3438 −4370 −4528 −3232 −5113 −2613 5320 −5052 — −147 −501232 42 −382 397 104 −625 209 −467 −722 276 — −3303 −3318 −325 −3473 −136−701 −1378 * *  3(F) −1308 −1104 −2227 −2120 3516 −2093 −244 −196 −189164 66 −1626 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −38−5840 −6882 −894 −1115 −943 −1060 * *  4(A) 1616 −1744 1125 33 −2015−1540 −262 −1686 937 −1765 −911 −252 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −901 −7402 −1125 −894 −1115 −2352 −314 * * 5(C) −346 2578 1084 −712 2092 −1540 −384 −167 −624 −482 125 −731 — −149−500 235 43 −381 398 106 −626 210 −466 −721 275 — −1009 −1006 −7567 −131−3527 −1916 −444 * *  6(S) 800 −586 −1937 −1415 −821 −1740 −954 1279−1204 −584 19 −1258 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −17 −6953 −7995 −894 −1115 −146 −3378 * *  7(K) −956 −2411 −803501 −2743 −1919 −558 −2483 2435 −2420 −1502 57 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  8(V) −2472 −2010 −5089 −4702 −2534 −4789 −4391 2241 −4574−151 −1318 −4442 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * *  9(Y) −4673 −3685 −5210 −55052423 −5069 −1332 −3424 −5065 −392 −2838 −3726 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  10(Y) −2170 −2625 −2489 −2097 −1555 −2986 −1481 −2628 906−2674 −2098 −2051 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * *  11(D) −2498 −4412 3500 1042−4581 −2437 −1765 −4500 733 −4361 −3682 515 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 12(K) 11 −2371 348 819 −2692 −535 −527 −2443 2294 −2387 −1461 590 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * *  13(D) −2663 −4633 3700 580 −4789 −2487 −1872−4738 731 −4578 −3963 −1073 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  14(C) 25033193 −4266 −3818 −2010 −3276 −2896 762 −3517 −1437 −1051 −3233 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  15(D) −1363 −2905 2748 542 −3202 −2072 −920 −2977290 −2912 −2023 1270 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  16(L) −1268 −1113 −3338−540 −1057 −2827 −1716 569 −2409 2299 −236 −2381 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  17(S) −1350 −2877 588 1045 −3189 −496 −920 −2963 −628 −2901−2011 1860 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−2336 −8139 −325 −894 −1115 −701 −1378 * *  18(G) −454 −832 −968 −1110−2112 3143 −1211 −2091 −1317 −2264 −1691 −978 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −38 −5840 −6882 −894 −1115 −3098−179 * *  19(H) −898 −1313 −545 −482 −320 −1336 4297 −1552 −160 −1493−1035 −579 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −38−5840 −6882 −894 −1115 −3098 −179 * *  20(D) −872 −1812 3234 432 −2215−967 −433 −2172 −569 −2269 −1704 99 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −38 −5840 −6882 −894 −1115 −3098 −179 * *  21(E)−766 −1695 521 2831 −2050 −1029 −293 −1804 −118 −1919 −1331 69 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −38 −5840 −6882 −894−1115 −3098 −179 * *  22(Y) −1337 −1229 −1681 −1596 1268 −1957 121 −918−1294 −769 −585 −1229 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −38 −5840 −6882 −894 −1115 −109 −3775 * *  23(I) −2294 −1931 −4749−4227 −1724 −4227 −3320 2306 −3952 1990 −634 −3878 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  24(I) −2801 −2299 −5406 −5003 −2108 −5164 −4649 3051 −48861593 −869 −4829 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * *  25(K) −234 −2632 306 −500−3007 −2141 −719 −2712 2540 −2619 −1730 −778 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 26(G) −2184 −3900 796 392 −4174 2903 −1580 −4030 −1636 −3937 −3173 −967— −149 −501 233 42 −375 399 104 −625 210 −463 −722 276 — −155 −3318−9181 −3674 −118 −701 −1378 * *  27(K) −3243 −3775 −4129 −2558 −4750−3647 −1490 −4021 3681 −3617 −2982 −2368 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  28(K)−1684 −2925 −1665 −979 −3407 −2535 −923 −3021 2737 −2865 −2032 202 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * *  29(V) −2623 −2122 −5300 −4990 −2769 −5101−5131 2388 −4945 −1532 −1474 −4790 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  30(A) 3309−1828 −4057 −4294 −4382 656 −3657 −4147 −4169 −4428 −3497 −2821 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  31(V) −2625 −2122 −5304 −4993 −2772 −5111 −51422881 −4950 −1532 −1474 −4796 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  32(I) −2790−2287 −5403 −5009 −2155 −5170 −4698 3324 −4899 1175 −912 −4835 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  33(G) −4435 −4203 −5092 −5462 −5893 3834 −5028−6627 −5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  34(Y) −4838−3766 −5229 −5579 1502 −5108 −1300 −3726 −5134 −3040 −3131 −3723 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  35(G) −4435 −4203 −5092 −5462 −5893 3834 −5028−6627 −5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  36(S) −1473−2007 −3647 −3780 −3430 −2363 −3314 228 −3616 −3373 −2876 −2840 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  37(Q) −4589 −4392 −3927 −4146 −5099 −4221 −4099−5973 −3840 −5564 −5304 −4230 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  38(G) 677−2128 −3838 −4171 −4647 3536 −3816 −4506 −4340 −4749 −3857 −3009 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  39(H) −2667 −3375 −2682 −2114 −3744 −3201 4738−3782 −445 −3553 −2886 −2112 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  40(A) 3631−2768 −4492 −4815 −4888 −2992 −4271 −4781 −4818 −5025 −4365 −3727 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  41(H) −3103 −3404 −2950 −2573 −783 −3679 4549−3407 −1372 −3071 −2715 −2454 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  42(A) 3357−1795 −4134 −4277 −4057 −2118 −3548 −3549 −4035 −4024 −3192 −2817 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  43(Q) −1061 1950 −2044 −1475 −1236 −2372 −1154−789 −1218 1062 1123 743 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  44(N) −4000 −4117−3389 −3749 −5073 −3911 −4123 −6022 −4503 −5797 −5419 4397 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  45(L) −4414 −3800 −5638 −5628 −2290 −4980 −4628 −1886−5423 3316 −1236 −5514 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  46(R) −1731 −3015275 −931 −3487 −2518 −973 −3116 2321 −2955 −2123 224 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  47(D) −2896 −4843 3855 944 −5037 −2600 −2082 −5082 −2528−4903 −4373 −1209 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * *  48(S) −1536 −2212 −2363−2679 −4293 −2279 −3082 −4365 −3331 −4524 −3676 288 — −148 −500 232 44−381 398 105 −627 211 −465 −721 275 — −155 −3318 −9181 −2405 −302 −701−1378 * *  49(G) −2521 −3968 1232 −911 −4849 3373 −2126 −4854 −2535−4752 −4136 −53 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * *  50(V) −2767 −2324 −5232 −4770396 −4827 −3784 −36 −4546 848 −611 −4472 — −148 −500 233 43 −381 399 106−626 211 −466 −720 275 — −148 −3381 −9181 −203 −2928 −701 −1378 * * 51(D) −1684 −3285 2735 2014 −3554 −2196 −1177 −3350 92 −3279 −2427 692— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * *  52(V) −3122 −2888 −5092 −5160 −3522 −4180−4687 −905 −5060 −2626 −2570 −4662 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  53(V) 369366 −3075 −2462 −883 −2557 −1420 1415 378 −757 −117 −2098 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  54(V) −2624 −2122 −5302 −4991 −2772 −5108 −5139 2623−4948 −1533 −1475 −4794 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  55(G) 929 −2107−3852 −4182 −4633 3492 −3809 −4486 −4335 −4732 −3835 −2997 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  56(L) −3427 −2938 −5791 −5325 −1449 −5374 −4410 −543−5063 3041 −255 −5207 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  57(R) −3040 −3724 −326682 −4620 −3470 −1396 −3905 804 −3529 −2874 −2133 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  58(K) 31 −2412 −803 1532 −2743 −1920 −559 −2483 1772 −2421−1503 −556 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * *  59(G) −2671 −4661 1614 587 −48323103 −1901 −4803 −2269 −4648 −4047 421 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  60(S)−1499 −2308 −1932 −1859 −4006 1604 −2121 −3754 1362 −3793 −2945 −1833 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −247 −8139 −2699−894 −1115 −701 −1378 * *  61(K) 1362 −2232 −619 −98 −2567 −427 −435−2309 1599 −2265 −1349 1101 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −9 −7900 −8943 −894 −1115 −344 −2238 * *  62(S) −1288−1904 −3742 −4011 −4384 −2155 −3593 −4209 −3996 −4479 −3573 −2789 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  63(W) 726 −873 −3261 −2634 1926 −2567 −1425 660−2252 −701 −68 −2174 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  64(E) 1527 −2404 2121636 −2722 −1878 −556 −2474 1241 −2419 −1497 350 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  65(K) −8 −2242 −895 770 −2502 −1963 −609 −2192 2589 22 −1353−631 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * *  66(A) 3631 −2768 −4492 −4815 −4888−2992 −4271 −4781 −4818 −5025 −4365 −3727 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 67(Q) −1006 −2441 −869 1767 −2780 −1965 −586 −2510 1702 −2445 −1534−603 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * *  68(A) 1489 −2393 167 1234 −2711 −1873−547 −2462 895 −2408 −1485 1161 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  69(D) 2104−2898 2124 985 −3163 −2096 1397 −2935 −693 −2897 −2025 −723 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  70(G) −2294 −2898 −2521 −2885 −4852 3641 −3456 −5042−3796 −5094 −4356 365 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  71(F) −2596 −2266 −4685−4188 3199 −4136 1018 505 −3812 1986 −405 −3595 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  72(K) 47 −2348 338 950 −2668 −1854 −512 −507 1721 −2364 −1438−490 — −149 −500 232 46 −381 399 105 −627 210 −466 −721 277 — −155 −3318−9181 −2159 −366 −701 −1378 * *  73(V) −1810 −1639 −4149 −3689 −1869−3417 −2822 29 −3369 320 −897 −3230 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  74(K) 847−1093 −2131 −1554 304 127 −1127 −637 1445 645 1186 −1534 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  75(T) −1284 −2794 1526 1290 −3096 −2041 1289 −2863 −548 −2808−1914 −668 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * *  76(V) −1089 −957 −3143 −2535 −943−2618 −1496 1052 −2198 −792 1859 −146 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  77(W)1606 −2321 −752 612 −2628 −323 −527 −2366 1480 −2331 −1416 −510 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * *  78(E) −1509 −3540 1372 3127 −3861 −120 −1391 −3685−1319 −3605 −2787 −900 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  79(A) 3390 −1868−4092 −4341 −4332 −2153 −3680 −3942 −4157 −4333 −3471 −2869 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  80(V) 2003 −1721 −4449 −3995 −2160 −3763 −3240 1342−3745 −1435 −1124 −3561 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  81(K) 1714 −2501−959 446 −2858 −2043 −654 −2574 1964 −2506 −1609 −689 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  82(W) 265 −2347 815 432 −2663 634 −519 −2410 619 −2361 −1438−495 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * *  83(A) 3391 −1860 −3998 −4279 −4411−2128 −3684 −4207 −4197 −4490 −3565 −2837 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 84(D) −2747 −4795 3813 396 −4912 −2496 −1935 −4905 −2324 −4735 −4166−1079 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * *  85(V) −2717 −2220 −5338 −4951 −2254−5099 −4670 1963 −4844 1553 −1011 −4759 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  86(V)−2635 −2129 −5306 −4970 −2652 −5125 −5011 2554 −4915 −354 −1368 −4781 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * *  87(M) −1340 −1208 −3317 −2708 −968 −2860−1708 577 −2346 932 4131 −2382 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  88(I) −2566−2177 −5017 −4470 669 −4496 −3487 2791 −4191 1116 1394 −4156 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * *  89(L) −4414 −3800 −5638 −5628 −2290 −4980 −4628 −1886−5423 3316 −1236 −5514 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  90(I) 1212 −1286−3846 −3262 −1360 −3195 −2166 1616 −2918 1031 −493 −2824 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  91(P) −1614 −2214 −3396 −3710 −4516 −2407 −3618 −4516 −3976−4705 −3849 −2890 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * *  92(D) −4580 −4701 4174 −3014−5700 −3967 −3905 −6376 −4478 −6024 −5744 −3355 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  93(E) −1123 −2199 −983 2715 −2589 −2046 −942 −2250 −625 −23561979 −870 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * *  94(H) 399 −1137 −2012 −14 1582−2306 1600 246 −1252 190 −325 −1456 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  95(Q)−2742 −3142 −2766 −2681 −2790 −3344 −2460 −160 −1802 −2456 −2353 −2682 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * *  96(A) 1981 −2315 −809 −268 −2645 −531 −579−2374 232 −2350 −1445 −567 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *  97(D) 491 −23511394 1381 −2671 −1854 1062 −2421 1010 −2367 −1440 −489 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * *  98(V) −2039 −1706 −4456 −3939 −1846 −3939 −3049 1986 −36561460 −826 804 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * *  99(Y) −4840 −3766 −5230 −55811898 −5109 −1300 −3727 −5135 −3041 −3132 −3723 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 100(E) −163 −2353 −734 1681 −2674 −1859 888 −2422 1668 −792−1443 777 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −257−8139 −2649 −894 −1115 −701 −1378 * * 101(E) 1017 −2763 862 2042 −3060−1913 −775 −2836 −495 −2773 −1886 1956 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −9 −7891 −8933 −894 −1115 −338 −2261 * * 102(E)−944 −2422 863 2138 −2740 −436 −567 −2493 894 −2437 −1515 −518 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 103(I) −2660 −2156 −5316 −4965 −2520 −5119 −49003165 −4894 297 −1251 −4775 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 104(E) 1068 −2341−760 2003 628 −1887 876 −2380 1240 −2347 −1436 −529 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 105(P) −343 −3144 1561 442 −3538 −489 −1216 −3329 −1038 −3274−2420 −848 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 106(N) −1173 −2375 −814 827 −2376−2071 1767 −2279 −479 −2336 −1509 3151 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 107(M)−3415 −2890 −5826 −5252 −1352 −5488 −4282 1361 −5022 2621 2728 −5181 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 108(K) −1941 −3098 −1997 −1232 −3650 −2740−1025 −3210 3059 −3010 −2208 499 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 109(P) 1129−2426 −740 964 −2747 −1913 −589 −2491 1139 −2440 −1525 −552 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 110(G) −2276 −2907 −2347 −2709 −4832 3554 −3349 −5005−3678 −5053 −4315 1193 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 111(A) 1730 −2349958 −198 −2661 −1868 −535 −2405 927 −2362 −1444 414 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 112(T) 1350 −1149 −14 −1461 −1155 −2314 −1111 758 −1275 −10241167 −1475 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 113(L) −3333 −2796 −5806 −5293−1506 −5535 −4502 1096 −5103 2935 −282 −5232 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *114(A) 1769 −1525 158 −857 −1603 148 −891 −1181 −752 187 −712 −1040 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 115(F) −4110 −3437 −5436 −5431 4216 −5143−2159 −1742 −5074 563 −1124 −4290 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 116(A) 3091−1829 −3998 −4219 −4413 119 −3637 −4216 −4134 −4469 −3523 −2798 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 117(H) −5197 −4539 −4720 −5009 −4036 −4506 5435−6314 −4911 −5786 −5667 −4954 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 118(G) −4435−4203 −5092 −5462 −5893 3834 −5028 −6627 −5765 −6297 −5970 −5141 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 119(F) −4044 −3387 −5534 −5444 4093 −5246 −2370−1514 −5107 1089 −868 −4443 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 120(N) 885−1899 −2020 −1781 −2956 −2135 −1925 −2602 −1809 3 −2052 3468 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 121(I) −2673 −2169 −5324 −4969 −2477 −5123 −4876 3293−4893 358 −1211 −4780 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 122(H) −3381 −3705 −3197−3491 −4166 638 5216 −5496 −3798 −5304 −4811 −3481 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 123(Y) −4816 −3757 −5210 −5549 3410 −5097 2153 −3719 −5105−3041 −3127 −3715 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 124(G) −1065 −2519 948 −272−2820 1844 998 −2566 972 −284 −1622 1553 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 125(Q)−412 −2285 −2466 −2186 −2068 −2877 −2019 −1589 −1588 1526 −1121 −2187 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 126(I) −2254 −1916 −4813 −4439 −2466 −4221−3932 3248 −4248 −1515 −1324 −4044 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 127(K) −888−2234 334 1172 −2504 −1881 −546 −93 1370 −300 −1337 465 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 128(P) 715 −1925 −3618 −3897 −4464 653 −3594 −4274 −4053 −4520−3596 −2770 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 129(P) 479 −2398 −1173 −637 −2915−2106 −848 −2610 −289 −2586 −1713 −884 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 130(A)1787 −2663 1377 529 −2976 −1992 −762 −2736 1785 −2680 −1776 −623 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −2336 −8139 −325 −894−1115 −701 −1378 * * 131(F) −1308 −1104 −2227 −2120 3516 −2093 −244 −196−1891 64 66 −1626 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −38 −5840 −6882 −894 −1115 −3098 −179 * * 132(P) −603 −937 −997 −1058−1832 −1041 −1092 −1737 −1074 −1874 −1416 −992 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −38 −5840 −6882 −894 −1115 −3098−179 * * 133(K) −804 −1483 −564 −230 −1920 −1335 −101 −1605 2889 −1630−1021 −349 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −38−5840 −6882 −894 −1115 −109 −3775 * * 134(D) −2405 −4159 3349 −651 −4260−261 −1744 −4307 −1947 −4207 −3514 2151 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 135(I)−2047 −1713 −4504 −3983 −1821 −3943 −3061 2461 −3697 1581 −797 −3593 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 136(D) −2024 −3444 3495 −680 −3868 −2331 −1596−44 −1632 −3675 −2915 685 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 137(V) −3122 −2888−5092 −5160 −3522 −4180 −4687 −905 −5060 −2626 −2570 −4662 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 138(I) −53 −875 −3230 −2609 1867 −393 −1422 2613 −2236−723 −81 −2157 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 139(M) 315 −2345 −4754 −4279−1396 −4001 −3301 −697 −3877 816 4676 −3879 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *140(V) −2623 −2122 −5301 −4990 −2770 −5102 −5132 2415 −4945 −1532 −1474−4791 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 141(A) 3405 2528 −4529 −4796 −4340 −2257−3851 −3901 −4447 −4351 −3532 −3057 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 142(P)−4853 −4392 −5213 −5573 −5853 −4408 −5077 −6679 −5780 −6281 −6067 −5357— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 143(K) −4484 −4357 −4380 −3992 −5413 −4236−3307 −5555 3994 −5171 −4707 −3921 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 144(G) 2167−1833 −3963 −4199 −4430 2715 −3642 −4236 −4146 −4489 −3540 −2795 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 145(P) −2604 −2948 −4094 −4235 −3544 −3269 −3767−3353 −3912 −3066 2095 −3659 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 146(G) −4435−4203 −5092 −5462 −5893 3834 −5028 −6627 −5765 −6297 −5970 −5141 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 147(H) −2569 −3440 −1867 −1702 −3820 −2996 4731−3830 634 −3639 −2963 −1838 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 148(T) 194−1498 −3255 −2899 −2240 −2226 −2291 −1754 −2652 1634 −1430 −2330 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 149(V) −3122 −2888 −5092 −5160 −3522 −4180 −4687−905 −5060 −2626 −2570 −4662 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 150(R) −4845−4446 −5107 −4682 −5507 −4412 −3791 −5946 −2789 −5502 −5118 −4521 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 151(R) −962 −2395 −777 1012 −2721 76 1031 −2459−142 −2413 −1501 −560 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 152(E) −902 −2032 −8992078 −2228 −1934 −611 −1897 −259 −221 −1156 520 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 153(Y) −4820 −3765 −5219 −5565 3303 −5093 −1317 −3703 −5127−3017 −3111 −3732 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 154(V) 129 −1901 −989 821−2060 −1969 −654 −1704 498 −52 −1037 −703 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *155(Q) 576 −2355 344 1156 −2675 −1856 −515 −508 1502 −2370 −1444 571 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 156(G) −3239 −3889 516 −2361 −5355 3646 −3337−5629 −3818 −5498 −4951 −2619 — −149 −500 232 44 −381 399 105 −627 211−466 −721 277 — −155 −3318 −9181 −2159 −366 −701 −1378 * * 157(G) 753−2516 −789 488 −2848 2300 −672 −2582 596 −2529 −1627 481 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 158(G) −52 −2212 −3792 −4133 −4698 3627 −3843 −4580 −4356−4812 −3937 −3058 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 159(V) −2485 −2030 −5123−4769 −2667 −4797 −4593 2349 −4661 −1545 −1424 −4502 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 160(P) −2541 −3139 −2413 −2753 −4726 −2991 −3342 −5055 −3527−5058 −4393 1199 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 161(C) 1577 3078 1357 −656−2664 −219 891 −2359 −617 −2434 −1576 −891 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *162(L) −2140 −2404 −3995 −3997 −2053 −3121 −3283 −1687 −3689 3041 −1200−3360 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 163(I) −2527 −2092 −5072 −4613 2047−4674 −3911 2668 −4413 117 −841 −4323 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 164(A)3631 −2768 −4492 −4815 −4888 −2992 −4271 −4781 −4818 −5025 −4365 −3727 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 165(V) −2623 −2122 −5301 −4990 −2770 −5102−5132 2426 −4946 −1532 −1474 −4791 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 166(H) −495−2631 903 −2051 722 −3242 3753 −2386 −2056 −2342 −1863 −2047 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 167(Q) −4589 −4392 −3927 −4146 −5099 −4221 −4099 −5973−3840 −5564 −5304 −4230 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 168(D) −2873 −46053943 −902 −4948 −2633 −2157 −5087 −2604 −4922 −4387 428 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 169(A) 1776 −1612 −1274 138 −1698 −2092 −816 −1295 150 −1526−780 −943 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 170(S) −1545 −2420 1001 −1518−4049 −2206 −2206 −3839 −2264 −3938 −3103 −1627 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 171(G) −2999 −3461 −2978 −3207 −5161 3669 −3454 −5283 15 −5188−4565 −3174 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 172(N) −887 −2349 −736 911 −2668−1860 −518 −2415 711 −2363 −1440 1880 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 173(A)3631 −2768 −4492 −4815 −4888 −2992 −4271 −4781 −4818 −5025 −4365 −3727 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 174(K) −1368 −2597 −1358 −720 −2953 −2298 1933−2603 1975 851 −1706 −958 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 175(D) −27 −26132320 1049 −2923 −1962 1973 −2684 544 −2624 −1712 1408 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 176(V) −1096 −938 −3279 −2658 −899 −2643 −1513 1265 1006 1388−124 −2232 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 177(A) 3342 −1826 −4064 −4295−4368 111 −3651 −4129 −4159 −4412 −3484 −2820 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 178(L) −4414 −3800 −5638 −5628 −2290 −4980 −4628 −1886 −54233316 −1236 −5514 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 179(S) 2216 −1831 −3961 −4157−4409 656 −3608 −4213 −4076 −4462 −3514 −2782 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 180(Y) −3634 −3050 −4918 −4872 36 −4597 −1405 223 −4437 −250−1998 −3545 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 181(A) 3391 −1860 −3998 −4279−4411 −2128 −3684 −4207 −4197 −4490 −3565 −2837 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 182(K) 370 307 113 −351 −2280 −1925 −615 324 1888 −2040 −1194−632 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 183(G) 2572 −2028 −3934 −4246 −4575 2752−3783 −4406 −4316 −4661 −3751 −2958 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 184(I)−2178 −1808 −4630 −4153 −2094 −4190 −3417 3121 −3909 311 −1023 698 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 185(G) −4435 −4203 −5092 −5462 −5893 3834−5028 −6627 −5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 186(G) 13922751 −4353 −4536 −4308 2864 −3681 −4084 −4233 −4354 −3425 −2859 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 187(G) 855 −1822 −3738 −3769 −4188 2507 −3358 −3950−3667 −4196 −3283 −2667 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 188(R) −3706 −3692−4490 −3846 1391 −4057 −2273 −3795 −1906 −3355 −3181 −3458 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −155 −8139 −3345 −894 −1115−701 −1378 * * 189(A) 2844 −1670 −3814 −3873 −4048 −1958 −3316 −3787−3686 −4061 −3156 −2598 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −7992 −9034 −894 −1115 −1303 −750 * * 190(G) −4176 −3995−4855 −5222 −5686 3828 −4823 −6386 −5533 −6087 −5741 −4896 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −7992 −9034 −894 −1115−422 −1980 * * 191(V) −2496 −2036 −5139 −4788 −2680 −4825 −4637 2522−4686 −1549 −1431 −4527 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 192(I) −2760 −2307−5270 −4785 1172 −4884 −3992 3346 −4576 662 −572 −4539 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 193(E) 454 −3086 −801 3279 −4125 −2346 −1868 −3919 −1868 −3932−3156 −1291 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 194(T) −2738 −3109 −4509 −4810−4918 −3305 −4346 −4865 −4769 −5072 −4551 −3987 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 195(T) −1323 −1975 −2766 −2978 −4207 −2152 −3105 −4004 −3295−4229 −3354 134 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 196(F) −4110 −3437 −5436 −54314216 −5143 −2159 −1742 −5074 563 −1124 −4290 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *197(K) −111 −2844 −1470 1220 −3294 −2415 −860 −2939 2448 −2798 −1945−1056 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 198(E) 545 −3735 1715 2880 −3981 −2308−1442 −3818 −1408 −3725 −2924 −909 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 199(E)−4574 −4665 −2714 3919 −5655 −3995 −3886 −6219 −4238 −5898 −5604 −3415 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 200(T) −1211 1331 −3446 −2962 −1399 −2495−1930 −869 −2610 −1374 −769 −2403 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 201(E)−1941 −3222 −921 3293 −3618 −2473 −1316 −3186 916 −3225 −2465 −1147 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 202(T) −1286 −1898 −3764 −4016 −4329 −2157−3572 −4120 −3959 −4408 −3517 −2789 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 203(D)−4580 −4701 4174 −3014 −5700 −3967 −3905 −6376 −4478 −6024 −5744 −3355 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 204(L) −3705 −3122 −6060 −5527 −1359 −5814−4569 1065 −5292 3069 −146 −5564 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 205(F)−3777 −3220 −5271 −5259 4268 −4892 −2120 417 −4916 −1143 −1314 −4142 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 206(G) −4435 −4203 −5092 −5462 −5893 3834−5028 −6627 −5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 207(E)−4574 −4665 −2714 3919 −5655 −3995 −3886 −6219 −4238 −5898 −5604 −3415 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 208(Q) −3157 −3746 −3170 −2450 −4497 −3515−1763 −4161 −443 −3809 −3189 −2392 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 209(A) 2672−1334 −3318 −2853 −1740 371 −2072 483 −2577 −1549 −928 −2359 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 210(V) −2620 −2125 −5293 −4983 −2756 −5076 −5100 1877−4932 −1522 −1466 −4777 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 211(L) −4414 −3800−5638 −5628 −2290 −4980 −4628 −1886 −5423 3316 −1236 −5514 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 212(C) −2243 5044 −4840 −4445 −1998 −3905 −3598 −31 −4138449 −930 −3902 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 213(G) −4435 −4203 −5092 −5462−5893 3834 −5028 −6627 −5765 −6297 −5970 −5141 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 214(G) 677 −2128 −3838 −4171 −4647 −3536 −3816 −4506 −4340−4749 −3857 −3009 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 215(V) 378 724 −3707 −3104−1180 −2986 −1919 1210 −2734 1302 −359 −2627 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *216(M) −948 −1407 −1515 156 −1452 −2164 1677 −1030 −821 −1302 1976 −1113— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 217(E) 1397 −2528 −725 2286 −2932 240 −791−2681 328 −2645 −1744 −674 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 218(L) −3705 −3122−6060 −5527 −1359 −5814 −4569 1065 −5292 3069 −146 −5564 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 219(V) −2600 −2108 −5251 −4894 −2568 −5025 −4783 2479 −4810−1354 1358 −4683 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 220(K) −1633 −2905 −1573 706−3375 −2487 −900 −3003 2925 −2849 −2008 −1128 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 221(A) 2352 2066 −2593 −2000 −947 −2434 −1271 −486 −32 −832714 −1817 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 222(G) 224 −1905 −3562 −3696 −36843361 −3297 −3220 −3625 81 −2886 −2733 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 223(F)−4781 −3756 −5207 −5542 4341 −5070 −1342 −3653 −5111 −2971 −3065 −3743 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 224(E) −2413 −4114 221 3465 −4392 −2485 −1689−4248 −1608 −4112 −3396 −1094 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 225(T) −1461−1864 −3139 −2645 −2659 −2483 −2136 −1734 −1646 −2359 −1761 −2298 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 226(L) −3831 −3266 −5314 −5148 −673 −5068 −2476−1443 −4706 3059 −789 −4359 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 227(V) −1819−1960 −4426 −4359 −2977 −3037 −3800 −439 −4098 −2145 −1909 −3451 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 228(E) −2863 −4790 1397 3563 −4990 −2594 −2061−5021 −2476 −4848 −4298 −1204 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 229(A) 2686−1916 −1440 275 −2529 −292 −1240 −2176 −998 −2345 1183 −1184 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 230(G) −4435 −4203 −5092 −5462 −5893 3834 −5028 −6627−5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 231(Y) −4099 −3483−4921 −5048 −109 −4705 −1565 −2914 −4494 −2334 2010 −3723 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 232(Q) 1711 −2410 −772 934 −2739 −1925 −604 −2477 −171−2433 −1524 −574 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 233(P) −3403 −4071 −1922 817−5220 −3359 −3173 −5423 −3337 −5281 −4771 −2564 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 234(E) −2870 −4786 1265 3587 −4993 −2600 −2068 −5026 −2483−4852 −4303 −1212 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 235(M) −3089 −2618 −5526−4976 −1443 −5128 −4045 653 −4735 1429 4269 −4803 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 236(A) 3631 −2768 −4492 −4815 −4888 −2992 −4271 −4781 −4818−5025 −4365 −3727 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 237(Y) −4797 −3764 −5203−5543 1114 −5069 −1339 −3694 −5111 −3013 −3107 −3741 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 238(F) −3828 −3605 −4146 −4086 4292 −4207 −2060 −3492 774−3071 −3005 −3556 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 239(E) −2775 −4471 −511 3582−4815 −2610 −2057 −4863 −2317 −4711 −4124 1234 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 240(C) −1407 5023 −4397 −4323 −3016 −2468 −3398 −1251 −3952−2540 −2082 −3044 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 241(L) −3370 −2847 −5795−5233 −1386 −5465 −4298 708 −5010 2859 1349 −5155 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 242(H) −2519 −4224 −445 946 −4287 −2505 4583 −4377 −1764 −4237−3571 2007 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 243(E) −3177 2571 −2701 3711 −4851−3438 −3479 −4765 −3558 −4932 −4406 −3081 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *244(L) −85 −1333 −3893 −3280 −1111 −3185 −2083 1066 −2910 2310 1961−2823 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 245(K) −2513 −3173 −2941 −2370 −4402−3094 −1824 −3895 3666 −3734 −3068 −2271 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −155 −3318 −9181 −196 −2974 −701 −1378 * *246(L) −3571 −3023 −5954 −5375 −1321 −5646 −4390 −632 −5138 2962 1671−5358 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 247(I) −2980 −2484 −5473 −5109 −1958−5196 −4587 3728 −4915 267 −781 −4933 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 248(V)−1685 −1668 −4095 −3732 −2081 −3082 −2893 −227 −3402 −1488 1383 −3123 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 249(D) −2963 −4569 3864 −1039 −4953 −2751−2187 −4998 767 −4822 −4260 −1424 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 250(L)−2768 −2715 −4842 −4633 −1675 −3998 −3790 −1038 −4207 3056 −562 −4150 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 251(M) −2822 −2356 −5342 −4861 −1759 −4985−4151 2587 −4663 173 4005 −4649 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 252(Y) −4562−3630 −5142 −5401 1516 −4992 −1300 −3544 −4968 −2963 −2986 −3671 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 253(E) −1959 −3457 −568 3135 −3841 −2347 −1410−3622 −1165 −3547 −2751 −1000 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 254(G) −347−2818 −1215 201 −3253 2635 −921 −2921 1474 −2822 −1972 −1002 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 255(G) −4435 −4203 −5092 −5462 −5893 3834 −5028 −6627−5765 −6297 −5970 −5141 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 256(I) −2042 −1769−4321 −3740 −1316 −3753 −2668 3134 −3389 1017 1999 194 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 257(A) 1914 −1640 −1237 128 −1748 −661 −793 −1355 −577 −49−817 −905 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 258(N) 365 −3809 1001 557 −40831196 −1518 −3930 −1535 −3838 −3055 3219 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 259(M)−3656 −3159 −5816 −5350 −1349 −5421 −4248 −822 −4928 948 4920 −5248 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 260(R) −1614 −1949 −2260 −1663 −886 −2765−1089 −1596 −1089 360 −1133 2239 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 261(Y)−1548 −2973 568 −509 −2846 −2207 1172 −2986 441 −2941 −2110 1645 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 262(S) 279 −1844 −3877 −4131 −4448 136 −3634 −4260−4132 −4511 −3561 −2782 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 263(I) −2653 −2149−5311 −4961 −2538 −5114 −4905 2957 −4891 332 −1267 −4770 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 264(S) −2212 −2711 −4019 −4348 −4697 −2899 −4045 −4988 −4527−5102 −4364 −3492 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 265(N) −2725 −4778 2906 990−4896 −2486 −1922 −4885 −2320 −4718 −4140 3045 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 266(T) −2061 −3396 −596 903 −4071 −2367 −1713 −3874 −1685−3859 −3103 2125 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 267(A) 3410 −2035 −3979 −4290−4573 659 −3798 −4400 −4332 −4660 −3754 −2978 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 268(E) −2118 −3486 −1036 2964 −3935 −2588 −1284 −3596 1878−3417 −2636 −1209 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 269(Y) −4524 −3618 −5100−5310 1910 −4972 −1299 −3522 634 −2951 −2965 −3649 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −155 −8139 −3345 −894 −1115 −701−1378 * * 270(G) −4176 −3995 −4855 −5222 −5686 3828 −4823 −6386 −5533−6087 −5741 −4896 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −7992 −9034 −894 −1115 −422 −1980 * * 271(D) −2710 −4705 3025 1828−4880 1758 −1932 −4863 −2320 −4703 −4115 −1084 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 272(Y) −2497 −2175 −4651 −4137 2447 −4046 −2215 255 −3766 8921558 −3537 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 273(V) −1425 −1250 −3480 −2894−1283 −3035 −1955 691 −2570 −1 −429 −2568 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *274(T) 516 −1643 −1918 −1401 −2170 −2112 −1387 −1759 −1234 −2016 −1265−1442 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 275(G) 677 −2128 −3838 −4171 −4647 3536−3816 −4506 −4340 −4749 −3857 −3009 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 276(P) −992−2210 343 −359 −2447 −1960 −675 −2143 533 −2204 −1351 −651 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 277(R) −1214 −2548 −1097 1072 175 −2145 −716 −2587 848−2528 −1653 −795 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 278(V) 289 −2035 −5133 −4789−2692 −4777 −4639 2142 −4689 −1561 −1443 −4511 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 279(I) −2265 −1919 −4828 −4452 −2473 −4254 −3954 3155 −4265−1516 −1326 −4066 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 280(D) −1731 −3162 2329 −550−3318 −2239 −1273 −3221 −1145 −3214 −2403 2295 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 281(E) 1097 −2699 1227 2368 −2994 −2011 −796 −2753 381 −2704−1808 −640 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 282(E) −166 −2372 859 1835 −2692−1861 1182 −2444 490 −2388 −1462 590 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 283(T)228 −1688 −3655 −3444 −3179 −2145 −2891 −2611 −3215 −3076 −2328 −2557 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 284(K) −2991 −3623 −3848 −2331 −4472 −3512−1356 −3763 2942 −3413 1860 −2188 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 285(E) 443−2370 732 1690 −2691 −1863 −526 −2442 1639 −2385 −1460 −498 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 286(A) 1871 −2286 −843 814 −2570 −1928 −578 269 1096−2279 −1391 −586 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 287(M) −3656 −3159 −5816−5350 −1349 −5421 −4248 −822 −4928 948 4920 −5248 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 288(K) −1646 −2891 −1591 287 −3346 −526 −912 −2971 2831 −2832−1997 −1146 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 289(E) −172 −2394 367 2205 −2713−487 −545 −2465 −134 −2409 −1485 1305 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 290(C)1574 3024 −4584 −4122 −2155 −3932 −3330 1746 −3870 −1406 −1109 −3691 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 291(L) −187 −2175 −4307 −3889 −898 −3779 −2344−944 −3485 2855 −476 −3390 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 292(K) 862 −2347 1431211 −2665 −1855 873 −2414 1692 −2362 917 −492 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 293(D) −2148 −3878 2790 1765 −4119 −2356 −1511 −3962 −1467−3852 −3075 24 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 294(I) −2630 −2131 −5302 −4991−2737 −5092 −5106 3464 −4941 −1495 −1447 −4789 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 295(Q) 346 −3134 −1818 −1401 −3862 −2760 −1314 −3433 1329−3271 −2513 −1545 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8139 −9181 −894 −1115 −701 −1378 * * 296(S) −1354 −2895 1712 354−3192 −2068 −914 −2967 −621 −2903 −2012 1817 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *297(G) −4435 −4203 −5092 −5462 −5893 3834 −5028 −6627 −5765 −6297 −5970−5141 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 298(E) −437 −2374 −769 2013 −2697 −1895−552 −2438 623 −2389 −1472 −536 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 299(F) −4347−3577 −4619 543 3858 −4820 −1320 −3438 −4609 −2900 −2894 −3532 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 300(A) 2827 −1603 −4068 −3628 −2047 −3165 −28231205 −3349 −1486 −1089 −3103 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 301(K) −2364−3363 −2464 163 −4038 −3041 −1188 −3501 2928 −3234 −2487 −1693 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 302(M) −893 −2361 740 1780 −2680 −537 −524 −2429930 −2376 1895 −498 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 303(W) −2965 −2553 −4795−4482 3045 −4315 −1779 −1426 −4093 92 −987 −3564 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 304(I) −2621 −2136 −5246 −4859 −2364 −4987 −4577 3052 −4747−250 1043 −4641 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 305(L) 684 −1319 −1741 761−1375 −2223 −1037 −927 −1042 1693 −533 −1287 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *306(E) 364 −4165 621 3314 −4393 −2398 −1686 −4282 −1836 −4169 −3446 728— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 307(N) −939 979 −1235 −681 −1738 1352 935−1357 −549 −1572 −816 2186 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 308(Q) 667 −2393−773 511 −2721 −316 −544 −2465 1211 −2405 −1484 584 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 309(A) 2050 −1857 −1081 −526 −2013 −2012 188 −1645 −385 −1213−1011 486 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 310(G) −1848 −2469 −2089 −22921262 2944 −2358 −3563 −2904 −3628 −3017 2347 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *311(Y) 475 1019 −1606 −1042 225 −2192 −935 −946 −891 −1226 1222 357 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 312(P) −87 −2372 −1362 756 −3738 −2205 −2007−3445 −1924 −3575 −2761 −1555 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −2336 −8139 −325 −894 −1115 −701 −1378 * * 313(K) −804−1483 −564 −230 −1920 −1335 −101 −1605 2889 −1630 −1021 −349 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −38 −5840 −6882 −894 −1115−3098 −179 * * 314(E) −766 −1695 521 2831 −2050 −1029 −293 −1804 −118−1919 −1331 69 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−38 −5840 −6882 −894 −1115 −109 −3775 * * 315(T) −942 −2382 −739 1086−2714 151 −581 −2459 −171 −2415 −1499 414 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *316(M) −2196 −1920 −4499 −3891 1726 −3822 −2504 −645 −3523 1973 3030−3442 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 317(H) −883 −2314 −747 517 −2618 −18631714 −647 1272 −2322 −1408 1011 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 318(A) 2474−2397 −816 −367 −2797 −273 −722 −2529 555 −2507 −1610 592 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115−701 −1378 * * 319(M) −154 −986 −2485 −337 1024 −375 −1232 325 −444 8671235 −1752 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 320(R) −1311 −2432 −1349 −724−2724 −2272 −799 −2361 613 −644 1079 976 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 321(R)897 −2364 −833 905 −2678 −1930 −568 −2405 1293 −2366 1485 −575 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 322(N) 505 −2300 −750 523 −2598 121 −525 −594 48595 −1395 1720 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 323(E) 444 −2266 −766 1488−2551 −1871 −533 −2276 889 −2266 1474 1478 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *324(N) 1511 −1770 −728 −204 −2244 −1781 −426 282 601 −2121 −1133 1769 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 325(N) 1053 −3109 1756 1735 −3404 −2143 −1074−3191 −846 −3124 −2254 2158 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 326(H) −2064−3071 −1245 −1267 −3262 −2570 4711 −3611 −1060 −3528 −2812 −1479 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894−1115 −701 −1378 * * 327(Q) −891 −2294 332 948 −2585 −739 −537 −2316−131 −138 −1391 −518 — −149 −500 233 43 −381 399 106 −626 210 −466 −720275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 328(I) −1632 −1661 −2846−86 −1626 −2983 −1663 3240 −1327 −191 −722 −2160 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 329(E) −2734 −3605 −1382 3593 −3624 −2986 −2317 −3317 −2175−3167 1983 −1898 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −2336 −8139 −325 −894 −1115 −701 −1378 * * 330(W) −1530 −1265 −2068−1964 479 −1810 −483 −1181 −1470 −968 −802 −1648 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −38 −5840 −6882 −894 −1115 −109−3775 * * 331(K) 8 −2067 −905 437 −2275 −1941 −611 −307 2031 −2031 −1189−636 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139−9181 −894 −1115 −701 −1378 * * 332(V) −2445 −2012 −5067 −4708 −2628−4682 −4459 1586 −4580 −1533 −1402 −4414 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 333(G)−4435 −4203 −5092 −5462 −5893 3834 −5028 −6627 −5765 −6297 −5970 −5141 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 334(E) 1447 −2744 −762 2410 −3114 −2113 −884−2850 −445 −2792 −1912 619 — −149 −500 233 43 −381 399 106 −626 210 −466−720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * * 335(K) −1204 −2643366 1309 −2998 −2086 −724 −2722 2626 −2637 −1741 −718 — −149 −500 233 43−381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 336(L) −3571 −3023 −5954 −5375 −1321 −5646 −4390 −632 −51382962 1671 −5358 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 —−8 −8139 −9181 −894 −1115 −701 −1378 * * 337(R) −4845 −4446 −5107 −4682−5507 −4412 −3791 −5946 −2789 −5502 −5118 −4521 — −149 −500 233 43 −381399 106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701−1378 * * 338(E) 943 −2422 1002 1200 −2741 377 −572 −2493 982 −2439−1517 −522 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8−8139 −9181 −894 −1115 −701 −1378 * * 339(M) −3391 −2886 −5774 −5202−1338 −5407 −4210 −576 −4943 1721 4369 −5109 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8139 −9181 −894 −1115 −701 −1378 * *340(M) −1812 −1601 −3997 −3375 696 −3401 −2222 −516 −2983 441 4360 −2991— −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −8 −8139 −9181−894 −1115 −701 −1378 * * 341(P) 102 −1789 −1729 −1313 428 −2112 −1410−2021 −1236 −2235 −1470 −1394 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −259 −8139 −2637 −894 −1115 −701 −1378 * * 342(W) −3486−3022 −4312 −4121 1891 −4228 −1173 −2749 310 −2389 −2250 −3147 — −149−500 233 43 −381 399 106 −626 210 −466 −720 275 — −239 −7889 −2749 −894−1115 −1590 −583 * * 343(I) −2220 −1737 −4860 −4531 −2271 −4617 −44973348 −4448 −1059 −1008 −4311 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −114 −7660 −3820 −894 −1115 −1149 −865 * * 344(A) 1699−2218 532 −33 −2555 −553 −413 −2304 1212 −2260 −1348 582 — −149 −500 23343 −381 399 106 −626 210 −466 −720 275 — −10 −7753 −8795 −894 −1115 −897−1111 * * 345(A) 1523 −2068 −769 −231 −2383 1040 −522 −2084 928 −2120−1245 −517 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −9−7949 −8991 −894 −1115 −1432 −668 * * 346(N) −1650 −3264 1760 −348 −3555−230 −1131 −3362 324 −3285 −2448 2847 — −149 −500 233 43 −381 399 106−626 210 −466 −720 275 — −193 −7842 −3049 −894 −1115 −1432 −668 * *347(K) 150 −2932 −2433 −1483 −3710 −2747 −907 −3141 3369 −2897 −2178−1487 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −11 −7660−8702 −894 −1115 −1824 −479 * * 348(L) −740 −922 −1768 154 −921 −2070−829 1384 247 1472 −100 −1202 — −149 −500 233 43 −381 399 106 −626 210−466 −720 275 — −11 −7660 −8702 −894 −1115 −943 −1059 * * 349(V) 138−1046 −3186 −2599 −1089 −2803 −1711 589 645 945 −236 −2305 — −149 −500233 43 −381 399 106 −626 210 −466 −720 275 — −9 −7842 −8885 −894 −1115−380 −2109 * * 350(D) −2086 −3722 2888 1158 −4001 1601 −1510 −3811 −1573−3773 −3018 −904 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275— −8 −8046 −9088 −894 −1115 −701 −1378 * * 351(K) −78 −2038 398 −137−2626 −483 542 −2374 2441 −2323 −1400 −447 — −149 −500 233 43 −381 399106 −626 210 −466 −720 275 — −8 −8046 −9088 −894 −1115 −701 −1378 * *352(D) 898 −1911 1604 −335 −2089 −1892 −573 −1746 489 −144 811 −609 —−149 −500 233 43 −381 399 106 −626 210 −466 −720 275 — −10 −7745 −8787−894 −1115 −701 −1378 * * 353(K) −1676 −2544 −1956 −1135 −2823 −2531−800 −2469 2955 −2426 −1683 −1240 — −149 −500 233 43 −381 399 106 −626210 −466 −720 275 — −11 −7649 −8691 −894 −1115 −701 −1378 * * 354(N)−1074 −1925 −1092 −1190 −3523 −1781 −1760 −3345 −1701 −3432 −2589 3429— * * * * * * * * * * * * — * * * * * * * * 0 Position in HMM P Q R S TV W Y alignment  1(Q) −1496 3263 122 −643 −684 −1239 −1542 −1030 7100% —394 45 96 359 117 −369 −294 −249 —  2(M) −4790 −4977 −4823 −4692 −4459−3629 −4103 −4017 7200% — 396 44 95 361 121 −368 −296 −251 —  3(F) −2278−1503 −1798 −1617 −1350 −389 305 1335 8600% — 394 45 96 359 117 −369−294 −249 —  4(A) −1658 154 −383 −488 640 −3 −2038 −1421 8700% — 394 4596 359 117 −369 −294 −249 —  5(C) −1705 −451 −883 −631 −338 −50 −774−133 8800% — 394 45 96 359 118 −369 −295 −249 —  6(S) −1964 −1013 −13581715 −476 1117 −1320 −938 9000% — 394 45 96 359 117 −369 −294 −249 — 7(K) −2010 1146 458 829 224 −2040 −2577 −1913 9100% — 394 45 96 359 117−369 −294 −249 —  8(V) −4600 −4417 −4628 −4080 −82 3023 −3952 −35109200% — 394 45 96 359 117 −369 −294 −249 —  9(Y) −4920 −3835 −4458 −4313−4533 −3643 −581 4349 9300% — 394 45 96 359 117 −369 −294 −249 —  10(Y)−3206 −1513 −1078 −2258 1039 −2435 −2009 4185 9400% — 394 45 96 359 117−369 −294 −249 —  11(D) −2961 −1429 −2799 −2158 −2558 −3974 −4550 −35419500% — 394 45 96 359 117 −369 −294 −249 —  12(K) −1960 −68 904 −67 −837−1993 −2554 −1871 9600% — 394 45 96 359 117 −369 −294 −249 —  13(D)−3046 −1551 −2987 −2292 −2742 −4201 −4759 −3709 9700% — 394 45 96 359117 −369 −294 −249 —  14(C) −3509 −3212 −3411 −2499 −1792 1507 −2796−2431 9800% — 394 45 96 359 117 −369 −294 −249 —  15(D) −2294 −489 −118653 1116 −2518 −3086 −2349 9900% — 394 45 96 359 117 −369 −294 −249 — 16(L) −2862 −2089 −2316 −232 −1213 1306 −1645 −1304 10000% — 394 45 96359 117 −369 −294 −249 —  17(S) −2289 −489 −1184 2139 190 −2503 −3077−2343 10100% — 394 45 96 359 117 −369 −294 −249 —  18(G) −1499 −1202−1421 −646 −774 −1550 −1916 −1919 10200% — 394 45 96 359 117 −369 −294−249 —  19(H) −1675 −363 −322 −934 −951 −1354 −725 107 10300% — 394 4596 359 117 −369 −294 −249 —  20(D) −1453 −184 −1141 −728 −973 −1814−2146 −1646 10400% — 394 45 96 359 117 −369 −294 −249 —  21(E) −1441 −4−527 −653 −814 −1512 −1988 −1505 10500% — 394 45 96 359 117 −369 −294−249 —  22(Y) −2163 −1111 −1301 −1443 −1359 −932 592 3932 10600% — 39445 96 359 117 −369 −294 −249 —  23(I) 94 −3538 −3812 −3411 −2247 1576−2891 −2629 10700% — 394 45 96 359 117 −369 −294 −249 —  24(I) −4788−4454 −4829 −4493 −2764 1435 −3781 −3585 10800% — 394 45 96 359 117 −369−294 −249 —  25(K) −2231 2257 968 −1109 −1152 −2288 −2738 −2136 10900% —394 45 96 359 117 −369 −294 −249 —  26(G) −2810 −1 −2362 1069 −2220−3530 −4130 −3229 11000% — 396 44 96 358 116 −371 −296 −251 —  27(K)−3580 −1076 1318 −3119 −2876 −3817 −3395 −3374 12600% — 394 45 96 359117 −369 −294 −249 —  28(K) −2582 1301 804 −1564 1681 −2645 −2905 −244812700% — 394 45 96 359 117 −369 −294 −249 —  29(V) −4868 −4890 −5101−4482 −2619 3219 −4505 −3990 12800% — 394 45 96 359 117 −369 −294 −249 — 30(A) −2904 −3694 −3937 −1470 59 −2957 −4610 −4522 12900% — 394 45 96359 117 −369 −294 −249 —  31(V) −4873 −4896 −5108 −4492 −2621 2896 −4512−3997 13000% — 394 45 96 359 117 −369 −294 −249 —  32(I) −4802 −4495−4860 −4506 −2757 1192 −3838 −3622 13100% — 394 45 96 359 117 −369 −294−249 —  33(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924 −5849 13200% —394 45 96 359 117 −369 −294 −249 —  34(Y) −4963 −3861 −4500 −4356 −4689−3881 2986 4507 13300% — 394 45 96 359 117 −369 −294 −249 —  35(G) −4804−5546 −5385 −4727 −4815 −5862 −4924 −5849 13400% — 394 45 96 359 117−369 −294 −249 —  36(S) −3093 −3395 −3541 3475 −1885 −2307 −3927 −347413500% — 394 45 96 359 117 −369 −294 −249 —  37(Q) −4693 4575 −3826−4704 −4772 −5612 −4577 −4751 13600% — 394 45 96 359 117 −369 −294 −249—  38(G) −3149 −3871 −4137 −1784 −2005 −3297 −4725 −4735 13700% — 394 4596 359 117 −369 −294 −249 —  39(H) 866 −1265 1506 −2614 −2557 −3469−3282 −2908 13800% — 394 45 96 359 117 −369 −294 −249 —  40(A) −3728−4477 −4545 −2567 −2762 −3852 −4724 −4942 13900% — 394 45 96 359 117−369 −294 −249 —  41(H) −3764 2546 −1428 −2990 −2976 −3308 2269 −29514000% — 394 45 96 359 117 −369 −294 −249 —  42(A) −2900 −3608 −3823 217−1660 −276 −4363 −4211 14100% — 394 45 96 359 117 −369 −294 −249 — 43(Q) −2446 2895 −1441 −1392 −1005 −693 −1678 −1278 14200% — 394 45 96359 117 −369 −294 −249 —  44(N) −4479 −4255 −4592 −4115 −4312 −5371−4650 −4731 14300% — 394 45 96 359 117 −369 −294 −249 —  45(L) −4997−4750 −5002 −5379 −4399 −2629 −3665 −3690 14400% — 394 45 96 359 117−369 −294 −249 —  46(R) −2603 256 2808 −1596 −1613 −2730 −2995 −251514500% — 394 45 96 359 117 −369 −294 −249 —  47(D) −3196 −1786 −3536−2501 −3007 −4517 −5004 −3956 14600% — 394 45 96 359 117 −369 −294 −249—  48(S) −3026 −2967 −3497 3508 −1962 −3259 −4477 −4066 14700% — 393 4595 360 118 −370 −295 −250 —  49(G) −3115 −1836 −3440 −2284 −2716 −4157−4880 −3914 15400% — 394 45 96 359 117 −369 −294 −249 —  50(V) −4518−3980 −4367 −4081 −2716 3323 −3037 −2660 15500% — 394 45 96 359 117 −369−294 −249 —  51(D) −2505 −770 −1595 −1483 −1666 332 −3460 −2676 15700% —394 45 96 359 117 −369 −294 −249 —  52(V) −4579 −4940 −4923 −4013 −32973796 −4414 −4190 15800% — 394 45 96 359 117 −369 −294 −249 —  53(V)−2610 −1809 −2037 −1630 1166 2145 −1385 −343 15900% — 394 45 96 359 117−369 −294 −249 —  54(V) −4871 −4894 −5106 −4488 −2620 3088 −4511 −399616000% — 394 45 96 359 117 −369 −294 −249 —  55(G) −3132 −3863 −4127−1761 −1982 −3275 −4720 −4725 16100% — 394 45 96 359 117 −369 −294 −249—  56(L) −4820 −4126 −4691 −4757 −3351 883 −3184 −3234 16200% — 394 4596 359 117 −369 −294 −249 —  57(R) −3439 −978 3800 −2894 −2709 −3682−3353 −3267 16300% — 394 45 96 359 117 −369 −294 −249 —  58(K) 1229 7271079 −566 −893 −2041 −2579 −1915 16400% — 394 45 96 359 117 −369 −294−249 —  59(G) −3049 −1587 −3230 −2297 −2766 −4245 −4850 −3752 16500% —394 45 96 359 117 −369 −294 −249 —  60(S) −2827 −1794 −1902 2738 −1771−2970 −3910 −3479 16600% — 394 45 96 359 117 −369 −294 −249 —  61(K)−1861 886 −512 833 −740 −1868 −2441 −1767 16700% — 394 45 96 359 117−369 −294 −249 —  62(S) −2948 −3606 −3832 3517 228 −3028 −4600 −445116800% — 394 45 96 359 117 −369 −294 −249 —  63(W) −2617 −1898 18 −1648−972 983 4091 −958 16900% — 394 45 96 359 117 −369 −294 −249 —  64(E)−1985 −100 −659 96 70 −2025 −2589 −1903 17000% — 394 45 96 359 117 −369−294 −249 —  65(K) −2052 692 −617 −889 −906 −361 −2455 −1836 17100% —394 45 96 359 117 −369 −294 −249 —  66(A) −3728 −4477 −4545 −2567 −2762−3852 −4724 −4942 17200% — 394 45 96 359 117 −369 −294 −249 —  67(Q)−2052 1923 873 −888 236 −630 −2596 −1949 17300% — 394 45 96 359 117 −369−294 −249 —  68(A) −1977 −90 −648 666 141 −2014 −2577 −1892 17400% — 39445 96 359 117 −369 −294 −249 —  69(D) −2329 −543 −1250 −1245 −1368 −2501−3087 −530 17500% — 394 45 96 359 117 −369 −294 −249 —  70(G) −3545−3376 −4005 −2451 −2700 −3996 −4706 −4575 17600% — 394 45 96 359 117−369 −294 −249 —  71(F) −3961 −3157 −3524 −3277 −2509 −1337 −1621 −84017700% — 394 45 96 359 117 −369 −294 −249 —  72(K) −1947 672 436 806 687−1970 −2533 −1851 17800% — 393 45 95 359 119 −370 −295 −250 —  73(V) 112−3099 −3291 −2619 −767 3269 −2708 −2354 18400% — 394 45 96 359 117 −369−294 −249 —  74(K) −2401 −1174 −1547 −764 −172 −528 −1519 1413 18500% —394 45 96 359 117 −369 −294 −249 —  75(T) −2242 −427 −1095 1451 1827−2411 −2986 −2264 18600% — 394 45 96 359 117 −369 −294 −249 —  76(V) 686−1884 −2111 −1695 945 2346 −1458 −1106 18700% — 394 45 96 359 117 −369−294 −249 —  77(W) −789 −73 421 23 −829 −1936 2212 −1843 18800% — 394 4596 359 117 −369 −294 −249 —  78(E) −2659 −1005 −1976 −400 −655 −3194−3790 −2957 18900% — 394 45 96 359 117 −369 −294 −249 —  79(A) −2948−3730 −3919 −1525 931 −2894 −4580 −4483 19000% — 394 45 96 359 117 −369−294 −249 —  80(V) −3855 −3494 −3700 −2979 −58 2574 −3091 −2698 19100% —394 45 96 359 117 −369 −294 −249 —  81(K) −2135 −203 1088 428 −1032−2148 −2652 −2027 19200% — 394 45 96 359 117 −369 −294 −249 —  82(W)−1952 1955 −609 −382 147 −1966 2858 −1853 19300% — 394 45 96 359 117−369 −294 −249 —  83(A) −2929 −3729 −3959 706 −1718 −3001 −4636 −453419400% — 394 45 96 359 117 −369 −294 −249 —  84(D) −3082 603 −3296 −2353−2844 −4347 −4929 −3809 19500% — 394 45 96 359 117 −369 −294 −249 — 85(V) −4771 −4509 −4836 −4427 −2688 2741 −3899 −3628 19600% — 394 45 96359 117 −369 −294 −249 —  86(V) −4852 −4798 −5038 −4487 −2622 3019 −4355−3902 19700% — 394 45 96 359 117 −369 −294 −249 —  87(M) −2878 250 −2265−228 −1278 −506 −1629 −1313 19800% — 394 45 96 359 117 −369 −294 −249 — 88(I) −4228 −3615 −3972 −3687 −2499 1692 −2860 −2711 19900% — 394 45 96359 117 −369 −294 −249 —  89(L) −4997 −4750 −5002 −5379 −4399 −2629−3665 −3690 20000% — 394 45 96 359 117 −369 −294 −249 —  90(I) −3211−2583 −2782 −2308 1598 1299 −2020 −1688 20100% — 394 45 96 359 117 −369−294 −249 —  91(P) 3993 −3625 −3900 666 −2068 −3354 −4610 −4474 20200% —394 45 96 359 117 −369 −294 −249 —  92(D) −4501 −3870 −4926 −4440 −4750−5894 −4922 −5231 20300% — 394 45 96 359 117 −369 −294 −249 —  93(E)−2250 −554 −1093 463 932 −1902 −2660 −2064 20400% — 394 45 96 359 117−369 −294 −249 —  94(H) −2374 1474 −1479 −94 −905 896 −1557 −1158 20500%— 394 45 96 359 117 −369 −294 −249 —  95(Q) −3710 4317 −1866 −2894 −2844−2559 −3295 −2711 20600% — 394 45 96 359 117 −369 −294 −249 —  96(A)1217 711 445 447 −874 −1951 −2540 −1883 20700% — 394 45 96 359 117 −369−294 −249 —  97(D) −1947 1017 362 −760 250 −623 −2535 −1852 20800% — 39445 96 359 117 −369 −294 −249 —  98(V) −3870 −3351 −3565 −3105 −2000 2330−2796 −2442 20900% — 394 45 96 359 117 −369 −294 −249 —  99(Y) −4964−3861 −4501 −4357 −4690 −3883 3325 4377 21000% — 394 45 96 359 117 −369−294 −249 — 100(E) −1952 890 286 −766 238 −1975 −2536 −1856 21100% — 39445 96 359 117 −369 −294 −249 — 101(E) −2143 −136 −1056 265 −1185 −2377−2948 −2207 21200% — 394 45 96 359 117 −369 −294 −249 — 102(E) −19941767 −673 109 −885 −1023 −2605 −1917 21300% — 394 45 96 359 117 −369−294 −249 — 103(I) −4828 −4705 −4975 −4470 −2642 2240 −4202 −3814 21400%— 394 45 96 359 117 −369 −294 −249 — 104(E) −1983 881 −618 −804 −855−1954 −2530 −1862 21500% — 394 45 96 359 117 −369 −294 −249 — 105(P)2974 −812 −1635 469 −1644 −2849 −3462 −2693 21600% — 394 45 96 359 117−369 −294 −249 — 106(N) −2218 −415 −957 −1093 −1120 −198 −2486 64721700% — 394 45 96 359 117 −369 −294 −249 — 107(M) −4778 −4005 −4613−4776 −3292 69 −3071 −3194 21800% — 394 45 96 359 117 −369 −294 −249 —108(K) −2766 1457 1261 −1817 −90 −2858 −3002 −2622 21900% — 394 45 96359 117 −369 −294 −249 — 109(P) 1941 1446 −655 −480 −913 −2050 −2610−1935 22000% — 394 45 96 359 117 −369 −294 −249 — 110(G) −3507 −3243−3937 −2418 −2674 −3974 −4703 −4521 22100% — 394 45 96 359 117 −369 −294−249 — 111(A) −1966 788 −630 790 −840 −303 −2540 −1863 22200% — 394 4596 359 117 −369 −294 −249 — 112(T) −2388 −1111 −1501 334 1843 354 −1581−1182 22300% — 394 45 96 359 117 −369 −294 −249 — 113(L) −4857 −4172−4762 −4864 −3236 506 −3264 −3351 22400% — 394 45 96 359 117 −369 −294−249 — 114(A) −2228 660 −1135 −1111 −913 1305 −1913 −1442 22500% — 39445 96 359 117 −369 −294 −249 — 115(F) −4871 −3987 −4561 −4547 −4016−2374 −1356 −292 22600% — 394 45 96 359 117 −369 −294 −249 — 116(A)−2896 −3656 −3927 1514 −1679 −2983 −4632 −4539 22700% — 394 45 96 359117 −369 −294 −249 — 117(H) −4960 −5011 −4732 −5391 −5395 −6022 −4063−3641 22800% — 394 45 96 359 117 −369 −294 −249 — 118(G) −4804 −5546−5385 −4727 −4815 −5862 −4924 −5849 22900% — 394 45 96 359 117 −369 −294−249 — 119(F) −4880 −3998 −4592 −4639 −3934 −2200 −1536 −523 23000% —394 45 96 359 117 −369 −294 −249 — 120(N) −2633 −1676 −2141 413 −1437−2139 −3194 −2737 23100% — 394 45 96 359 117 −369 −294 −249 — 121(I)−4824 −4681 −4961 −4472 −2653 1969 −4158 −3791 23200% — 394 45 96 359117 −369 −294 −249 — 122(H) −4185 −3770 −3879 −3508 −3702 −4793 −4170−3759 23300% — 394 45 96 359 117 −369 −294 −249 — 123(Y) −4955 −3851−4483 −4344 −4669 −3870 −547 3677 23400% — 394 45 96 359 117 −369 −294−249 — 124(G) −2090 −229 −802 −938 −1011 −2133 −2708 −2021 23500% — 39445 96 359 117 −369 −294 −249 — 125(Q) −3153 3585 −1718 −2137 −1964 −1719−2789 −2414 23600% — 394 45 96 359 117 −369 −294 −249 — 126(I) −4259−4063 −4255 230 −2280 2003 −3673 −3237 23700% — 394 45 96 359 117 −369−294 −249 — 127(K) −1974 655 −646 −794 −827 1255 −2448 −1798 23800% —394 45 96 359 117 −369 −294 −249 — 128(P) 3775 −3593 −3911 −1550 −1770−3067 −4647 −4548 23900% — 394 45 96 359 117 −369 −294 −249 — 129(P)2238 1247 2195 51 −1147 −2184 −2757 −2174 24000% — 394 45 96 359 117−369 −294 −249 — 130(A) −2161 −319 −936 297 −1120 −2285 −2853 −214624100% — 394 45 96 359 117 −369 −294 −249 — 131(F) −2278 −1503 −1798−1617 −1350 −389 305 1335 24200% — 394 45 96 359 117 −369 −294 −249 —132(P) 3539 −1065 −1192 −789 −866 −1383 −1765 −1661 24300% — 394 45 96359 117 −369 −294 −249 — 133(K) −1569 232 698 −786 −759 −1358 −1637−1317 24400% — 394 45 96 359 117 −369 −294 −249 — 134(D) −2936 −1416−2754 −2102 −2471 −3802 −4324 637 24500% — 394 45 96 359 117 −369 −294−249 — 135(I) −3873 −3371 −3587 −342 −2009 1904 −2784 −2441 24600% — 39445 96 359 117 −369 −294 −249 — 136(D) −2782 −1248 −2305 478 −2088 −3196−3911 −3098 24700% — 394 45 96 359 117 −369 −294 −249 — 137(V) −4579−4940 −4923 −4013 −3297 3796 −4414 −4190 24800% — 394 45 96 359 117 −369−294 −249 — 138(I) −2608 −1885 −2086 −1633 276 −271 −1325 844 24900% —394 45 96 359 117 −369 −294 −249 — 139(M) −3994 −3361 −3676 −3242 −2531−1114 −2746 −2629 25000% — 394 45 96 359 117 −369 −294 −249 — 140(V)−4869 −4890 −5102 −4483 −2619 3206 −4506 −3991 25100% — 394 45 96 359117 −369 −294 −249 — 141(A) −3052 −3976 −4112 −1643 −1844 −2929 −4572−4519 25200% — 394 45 96 359 117 −369 −294 −249 — 142(P) 4310 −5648−5396 −5166 −5194 −6092 −4900 −5786 25300% — 394 45 96 359 117 −369 −294−249 — 143(K) −4535 −3079 −2169 −4529 −4408 −5264 −4403 −4729 25400% —394 45 96 359 117 −369 −294 −249 — 144(G) −2898 −3661 −3939 910 −1682−2994 −4647 −4556 25500% — 394 45 96 359 117 −369 −294 −249 — 145(P)4036 −3912 −3822 −2883 −2963 −3249 −4027 −3787 25600% — 394 45 96 359117 −369 −294 −249 — 146(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924−5849 25700% — 394 45 96 359 117 −369 −294 −249 — 147(H) 1551 −1305 −748−2470 −2510 −3482 −3434 −2990 25800% — 394 45 96 359 117 −369 −294 −249— 148(T) −2747 −2399 −2684 567 2687 −1484 −2682 −2351 25900% — 394 45 96359 117 −369 −294 −249 — 149(V) −4579 −4940 −4923 −4013 −3297 3796 −4414−4190 26000% — 394 45 96 359 117 −369 −294 −249 — 150(R) −4754 −36724219 −4989 −4832 −5644 −4538 −4993 26100% — 394 45 96 359 117 −369 −294−249 — 151(R) −2018 −128 2308 1224 −66 −2023 −2585 −1919 26200% — 394 4596 359 117 −369 −294 −249 — 152(E) −2024 816 −736 −858 1303 −287 −2295537 26300% — 394 45 96 359 117 −369 −294 −249 — 153(Y) −4959 −3868 −4500−4356 −4679 −3867 −565 4052 26400% — 394 45 96 359 117 −369 −294 −249 —154(V) −2057 695 −796 443 −344 1871 −2192 −1626 26500% — 394 45 96 359117 −369 −294 −249 — 155(Q) −1949 1878 419 −764 −822 −1976 −2538 −185626600% — 394 45 96 359 117 −369 −294 −249 — 156(G) −3905 −3187 −4377−3211 −3532 −4837 −4895 −4826 26700% — 393 45 95 359 117 −368 −295 −250— 157(G) −2112 −224 471 −962 −1024 −2149 −2694 −2033 27300% — 394 45 96359 117 −369 −294 −249 — 158(G) −3216 −3901 −4170 −1874 −2095 −3384−4734 −4766 27400% — 394 45 96 359 117 −369 −294 −249 — 159(V) −4648−4554 −4752 −4115 825 2986 −4159 −3678 27500% — 394 45 96 359 117 −369−294 −249 — 160(P) 4031 −3244 −3757 −2665 −2911 −4148 −4583 −4362 27600%— 394 45 96 359 117 −369 −294 −249 — 161(C) −2199 −545 −1093 872 −1043−1946 −2701 −2102 27700% — 394 45 96 359 117 −369 −294 −249 — 162(L)−3626 −3433 −3567 480 −2414 −1973 −3145 −2755 27800% — 394 45 96 359 117−369 −294 −249 — 163(I) −4439 −4023 −4320 −3916 −2488 2176 −3342 −304027900% — 394 45 96 359 117 −369 −294 −249 — 164(A) −3728 −4477 −4545−2567 −2762 −3852 −4724 −4942 28000% — 394 45 96 359 117 −369 −294 −249— 165(V) −4869 −4891 −5102 −4483 −2619 3200 −4506 −3991 28100% — 394 4596 359 117 −369 −294 −249 — 166(H) −3330 −1815 −2362 −2318 −2233 −2272−981 3315 28200% — 394 45 96 359 117 −369 −294 −249 — 167(Q) −4693 4575−3826 −4704 −4772 −5612 −4577 −4751 28300% — 394 45 96 359 117 −369 −294−249 — 168(D) −3235 −1872 −3575 −2522 −3009 −4491 −4932 −3946 28400% —394 45 96 359 117 −369 −294 −249 — 169(A) 1212 −538 −1013 −1056 −8941275 −1968 635 28500% — 394 45 96 359 117 −369 −294 −249 — 170(S) −2814−1909 −2758 2666 2313 −3045 −4143 −3560 28600% — 394 45 96 359 117 −369−294 −249 — 171(G) −3946 −3312 −3140 −3128 −3317 −4492 −4622 −474928700% — 394 45 96 359 117 −369 −294 −249 — 172(N) −1954 436 799 6711230 −539 −2534 −1855 28800% — 394 45 96 359 117 −369 −294 −249 — 173(A)−3728 −4477 −4545 −2567 −2762 −3852 −4724 −4942 28900% — 394 45 96 359117 −369 −294 −249 — 174(K) −2360 859 1012 −1273 1094 −2245 −2690 −216529000% — 394 45 96 359 117 −369 −294 −249 — 175(D) −2115 345 −869 −969−1058 −2231 −2793 −2087 29100% — 394 45 96 359 117 −369 −294 −249 —176(V) −2688 −1950 −2151 −1725 −311 1669 −1425 827 29200% — 394 45 96359 117 −369 −294 −249 — 177(A) −2903 −3689 −3929 −1469 409 −2949 −4599−4508 29300% — 394 45 96 359 117 −369 −294 −249 — 178(L) −4997 −4750−5002 −5379 −4399 −2629 −3665 −3690 29400% — 394 45 96 359 117 −369 −294−249 — 179(S) −2892 −3613 −3895 2686 −1675 −2982 −4623 −4523 29500% —394 45 96 359 117 −369 −294 −249 — 180(Y) −4494 −3522 −4004 −3782 −3536−2621 2928 4349 29600% — 394 45 96 359 117 −369 −294 −249 — 181(A) −2929−3729 −3959 706 −1718 −3001 −4636 −4534 29700% — 394 45 96 359 117 −369−294 −249 — 182(K) −2022 −205 −736 1541 14 −1628 −2330 −1726 29800% —394 45 96 359 117 −369 −294 −249 — 183(G) −3070 −3837 −4092 −1679 −1898−3191 −4701 −4686 29900% — 394 45 96 359 117 −369 −294 −249 — 184(I)−4099 −3656 −3864 −3388 −2148 1742 −3147 −2761 30000% — 394 45 96 359117 −369 −294 −249 — 185(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924−5849 30100% — 394 45 96 359 117 −369 −294 −249 — 186(G) −2890 −3744−3957 712 −1656 −2914 −4553 −4470 30200% — 394 45 96 359 117 −369 −294−249 — 187(G) 986 −3302 −3621 −1441 2334 −2867 −4408 −4236 30300% — 39445 96 359 117 −369 −294 −249 — 188(R) −4263 −2675 3948 −3768 −3671 −3813−2293 −1328 30400% — 394 45 96 359 117 −369 −294 −249 — 189(A) −2734−3301 −3572 1088 1907 −2713 −4286 −4136 30500% — 394 45 96 359 117 −369−294 −249 — 190(G) −4606 −5312 −5178 −4461 −4560 −5613 −4754 −563530600% — 394 45 96 359 117 −369 −294 −249 — 191(V) −4668 −4584 −4783−4148 629 2919 −4191 −3706 30700% — 394 45 96 359 117 −369 −294 −249 —192(I) −4535 −4008 −4400 −4134 −2703 757 −3223 −3041 30800% — 394 45 96359 117 −369 −294 −249 — 193(E) 1208 −1533 −2450 −1842 −2097 −3305 −4121−3395 30900% — 394 45 96 359 117 −369 −294 −249 — 194(T) −3998 −4580−4545 −2999 4033 −4113 −4684 −4915 31000% — 394 45 96 359 117 −369 −294−249 — 195(T) −2901 −2988 −3387 −113 3742 −2969 −4405 −4119 31100% — 39445 96 359 117 −369 −294 −249 — 196(F) −4871 −3987 −4561 −4547 −4016−2374 −1356 −292 31200% — 394 45 96 359 117 −369 −294 −249 — 197(K)−2475 767 2054 −1421 −1432 −2544 −2858 −2356 31300% — 394 45 96 359 117−369 −294 −249 — 198(E) −2713 1281 −2087 −1777 −2041 −3331 −3909 −304631400% — 394 45 96 359 117 −369 −294 −249 — 199(E) −4513 −3838 −4570−4456 −4726 −5786 −4878 −5197 31500% — 394 45 96 359 117 −369 −294 −249— 200(T) −2815 −2309 −2526 −1685 3305 195 −1933 1430 31600% — 394 45 96359 117 −369 −294 −249 — 201(E) −2749 −923 −1064 −1790 −1902 −171 −3398−2802 31700% — 394 45 96 359 117 −369 −294 −249 — 202(T) −2947 −3588−3797 697 3756 −2989 −4554 −4399 31800% — 394 45 96 359 117 −369 −294−249 — 203(D) −4501 −3870 −4926 −4440 −4750 −5894 −4922 −5231 31900% —394 45 96 359 117 −369 −294 −249 — 204(L) −4963 −4163 −4828 −5215 −3571−1279 −3159 −3298 32000% — 394 45 96 359 117 −369 −294 −249 — 205(F)−4753 −3956 −4473 −4270 −3740 −1899 −1349 −269 32100% — 394 45 96 359117 −369 −294 −249 — 206(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924−5849 32200% — 394 45 96 359 117 −369 −294 −249 — 207(E) −4513 −3838−4570 −4456 −4726 −5786 −4878 −5197 32300% — 394 45 96 359 117 −369 −294−249 — 208(Q) −3620 4200 1284 −3063 −2944 −3900 −3556 −3420 32400% — 39445 96 359 117 −369 −294 −249 — 209(A) −2798 −2295 −2567 −1629 191 932−2245 −1899 32500% — 394 45 96 359 117 −369 −294 −249 — 210(V) −4855−4870 −5082 −4456 −2619 3416 −4480 −3969 32600% — 394 45 96 359 117 −369−294 −249 — 211(L) −4997 −4750 −5002 −5379 −4399 −2629 −3665 −369032700% — 394 45 96 359 117 −369 −294 −249 — 212(C) −4040 −3778 −4010−3184 −2306 1347 −3209 −2883 32800% — 394 45 96 359 117 −369 −294 −249 —213(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924 −5849 32900% — 394 4596 359 117 −369 −294 −249 — 214(G) −3149 −3871 −4137 −1784 −2005 −3297−4725 −4735 33000% — 394 45 96 359 117 −369 −294 −249 — 215(V) −3014−2382 −2566 −2089 1123 1949 −1773 −1423 33100% — 394 45 96 359 117 −369−294 −249 — 216(M) −2245 −718 −1173 773 1715 1332 −1794 −1343 33200% —394 45 96 359 117 −369 −294 −249 — 217(E) −2162 −351 −939 545 −1095−2227 −2828 −2143 33300% — 394 45 96 359 117 −369 −294 −249 — 218(L)−4963 −4163 −4828 −5215 −3571 −1279 −3159 −3298 33400% — 394 45 96 359117 −369 −294 −249 — 219(V) −4772 −4654 −4895 −4362 −2584 3018 −4181−3758 33500% — 394 45 96 359 117 −369 −294 −249 — 220(K) −2541 1714 784−1509 −105 −2617 −2894 −2418 33600% — 394 45 96 359 117 −369 −294 −249 —221(A) −2498 −1493 −1792 −1483 274 453 −1419 501 33700% — 394 45 96 359117 −369 −294 −249 — 222(G) −2977 −3326 −3545 −1606 −1763 −2574 −4068−3810 33800% — 394 45 96 359 117 −369 −294 −249 — 223(F) −4949 −3874−4496 −4351 −4650 −3829 −591 1725 33900% — 394 45 96 359 117 −369 −294−249 — 224(E) −2951 −1336 871 −2119 −2441 −3763 −4239 −3395 34000% — 39445 96 359 117 −369 −294 −249 — 225(T) −2936 −1995 920 −1748 3354 967−2989 −2654 34100% — 394 45 96 359 117 −369 −294 −249 — 226(L) −4756−3864 −4314 −4462 −3729 −2115 −1672 1736 34200% — 394 45 96 359 117 −369−294 −249 — 227(V) −3600 −3879 −4011 −2397 2510 2999 −3974 −3608 34300%— 394 45 96 359 117 −369 −294 −249 — 228(E) −3182 −1761 −3454 −2476−2968 −4462 −4951 −3920 34400% — 394 45 96 359 117 −369 −294 −249 —229(A) −2355 −905 −1425 512 −1158 −1817 −2697 −2174 34500% — 394 45 96359 117 −369 −294 −249 — 230(G) −4804 −5546 −5385 −4727 −4815 −5862−4924 −5849 34600% — 394 45 96 359 117 −369 −294 −249 — 231(Y) −4707−3735 −4111 −4065 −4068 −3172 −847 4618 34700% — 394 45 96 359 117 −369−294 −249 — 232(Q) −2035 2086 345 902 −923 −2042 −2608 −1941 34800% —394 45 96 359 117 −369 −294 −249 — 233(P) 4045 −2989 −3760 −3320 −3624−4817 −4763 −4636 34900% — 394 45 96 359 117 −369 −294 −249 — 234(E)−3188 −1768 −3458 −2484 −2974 −4467 −4950 −3925 35000% — 394 45 96 359117 −369 −294 −249 — 235(M) −4610 −3915 −4423 −4378 −2995 1140 −3054−3074 35100% — 394 45 96 359 117 −369 −294 −249 — 236(A) −3728 −4477−4545 −2567 −2762 −3852 −4724 −4942 35200% — 394 45 96 359 117 −369 −294−249 — 237(Y) −4951 −3876 −4497 −4354 −4666 −3859 −588 4723 35300% — 39445 96 359 117 −369 −294 −249 — 238(F) −4434 −3287 −2952 −3868 −3858−3571 −1593 −494 35400% — 394 45 96 359 117 −369 −294 −249 — 239(E)−3182 −1755 −3103 −2442 −2884 −4306 −4753 −3820 35500% — 394 45 96 359117 −369 −294 −249 — 240(C) −3133 −3588 −3744 −1803 1473 1125 −3677−3390 35600% — 394 45 96 359 117 −369 −294 −249 — 241(L) −4777 −4026−4622 −4755 −3254 814 −3103 −3213 35700% — 394 45 96 359 117 −369 −294−249 — 242(H) −3009 −1439 −2361 −2209 −2569 −3893 −4267 −3353 35800% —394 45 96 359 117 −369 −294 −249 — 243(E) −4005 −3370 −3802 −3269 −3451−4260 −4554 −4524 35900% — 394 45 96 359 117 −369 −294 −249 — 244(L)−3170 −2501 −2721 −2289 −113 436 −1859 −1558 36000% — 394 45 96 359 117−369 −294 −249 — 245(K) −3377 −1447 −616 −2537 751 −3485 −3625 −347136100% — 394 45 96 359 117 −369 −294 −249 — 246(L) −4852 −4044 −4689−4963 −3436 742 −3082 −3239 36300% — 394 45 96 359 117 −369 −294 −249 —247(I) −4833 −4427 −4799 −4598 −2949 −64 −3627 −3397 36400% — 394 45 96359 117 −369 −294 −249 — 248(V) −3419 −3145 −3320 367 −1807 3332 −2874−2504 36500% — 394 45 96 359 117 −369 −294 −249 — 249(D) −3314 −1891−3072 −2624 −3060 −4467 −4770 −3962 36600% — 394 45 96 359 117 −369 −294−249 — 250(L) −4179 −3740 −3989 −3399 699 −1545 −3154 −3367 36700% — 39445 96 359 117 −369 −294 −249 — 251(M) −4601 −4076 −4487 −4251 −2764 766−3321 −3210 36800% — 394 45 96 359 117 −369 −294 −249 — 252(Y) −4868−3786 −4393 381 −4432 −3662 2413 4375 36900% — 394 45 96 359 117 −369−294 −249 — 253(E) −2716 1879 −1666 −1757 692 −3162 −3709 −2960 37000% —394 45 96 359 117 −369 −294 −249 — 254(G) −2459 −486 658 −1397 −1435−2521 −2923 −2378 37100% — 394 45 96 359 117 −369 −294 −249 — 255(G)−4804 −5546 −5385 −4727 −4815 −5862 −4924 −5849 37200% — 394 45 96 359117 −369 −294 −249 — 256(I) −3653 −2958 −3221 −2884 −1980 −344 −2325−2073 37300% — 394 45 96 359 117 −369 −294 −249 — 257(A) −2155 −498 −9931037 162 −1149 −2002 624 37400% — 394 45 96 359 117 −369 −294 −249 —258(N) −2763 −1148 −2243 −1845 −2131 −3433 −4027 −3144 37500% — 394 4596 359 117 −369 −294 −249 — 259(M) −4838 −4039 −4539 −4860 −3558 −1557−3044 −3030 37600% — 394 45 96 359 117 −369 −294 −249 — 260(R) −2814−1215 2408 −1789 −1535 −1468 1995 1546 37700% — 394 45 96 359 117 −369−294 −249 — 261(Y) −2446 −659 −1264 −1389 −1509 −2582 −2924 3695 37800%— 394 45 96 359 117 −369 −294 −249 — 262(S) −2903 −3648 −3936 3391 −1692−3009 −4661 −4566 37900% — 394 45 96 359 117 −369 −294 −249 — 263(I)−4827 −4713 −4978 −4466 −2636 2521 −4218 −3821 38000% — 394 45 96 359117 −369 −294 −249 — 264(S) −3638 −4203 −4355 3681 −2664 −3902 −4616−4605 38100% — 394 45 96 359 117 −369 −294 −249 — 265(N) −3069 −1612−3311 −2334 −2821 −4325 −4919 −3794 38200% — 394 45 96 359 117 −369 −294−249 — 266(T) −2850 −1369 −2277 −1897 3157 −3351 −4055 −3271 38300% —394 45 96 359 117 −369 −294 −249 — 267(A) −3080 −3858 −4099 −1690 −1908−3194 −4697 −4687 38400% — 394 45 96 359 117 −369 −294 −249 — 268(E)−2827 1323 −773 −1923 −2032 −3199 −3455 −2917 38500% — 394 45 96 359 117−369 −294 −249 — 269(Y) −4847 −3741 −4299 −4199 −4391 −3637 2997 421138600% — 394 45 96 359 117 −369 −294 −249 — 270(G) −4606 −5312 −5178−4461 −4560 −5613 −4754 −5635 38700% — 394 45 96 359 117 −369 −294 −249— 271(D) −3073 −1621 −3297 −2330 −2809 −4301 −4894 −3793 38800% — 394 4596 359 117 −369 −294 −249 — 272(Y) −3886 −3109 −3468 −3181 −2410 −1282−1615 3508 38900% — 394 45 96 359 117 −369 −294 −249 — 273(V) −3060 942−2514 −2129 1701 2578 −1907 −1553 39000% — 394 45 96 359 117 −369 −294−249 — 274(T) −2421 −1149 887 1341 2345 822 −2454 −2006 39100% — 394 4596 359 117 −369 −294 −249 — 275(G) −3149 −3871 −4137 −1784 −2005 −3297−4725 −4735 39200% — 394 45 96 359 117 −369 −294 −249 — 276(P) 2813 −260−802 465 −939 −873 −2467 1093 39300% — 394 45 96 359 117 −369 −294 −249— 277(R) −2228 −273 2862 417 −1133 −2191 −2671 −2084 39400% — 394 45 96359 117 −369 −294 −249 — 278(V) −4649 −4585 −4784 −4102 −2487 3125 −4202−3717 39500% — 394 45 96 359 117 −369 −294 −249 — 279(I) −4279 −4082−4274 226 −2288 2182 −3688 −3250 39600% — 394 45 96 359 117 −369 −294−249 — 280(D) −2573 −899 −1742 −1561 1851 −2804 −3366 1327 39700% — 39445 96 359 117 −369 −294 −249 — 281(E) −2190 −358 −992 −1065 −1162 −2310−2885 1152 39800% — 394 45 96 359 117 −369 −294 −249 — 282(E) 478 1356−620 116 −837 −1994 −2555 −1871 39900% — 394 45 96 359 117 −369 −294−249 — 283(T) −2821 −2916 −3214 2251 2366 1397 −3549 −3270 40000% — 39445 96 359 117 −369 −294 −249 — 284(K) −3430 −937 2705 −2874 −2650 −3556−3267 −3189 40100% — 394 45 96 359 117 −369 −294 −249 — 285(E) −19601120 437 106 −836 −1992 −2552 −1869 40200% — 394 45 96 359 117 −369 −294−249 — 286(A) −2019 −136 1056 −62 −881 −1889 −2488 −1849 40300% — 394 4596 359 117 −369 −294 −249 — 287(M) −4838 −4039 −4539 −4860 −3558 −1557−3044 −3030 40400% — 394 45 96 359 117 −369 −294 −249 — 288(K) −2554−472 1762 −1527 −1524 −2597 −2885 1245 40500% — 394 45 96 359 117 −369−294 −249 — 289(E) −1975 663 831 −795 72 −2015 −2577 −1891 40600% — 39445 96 359 117 −369 −294 −249 — 290(C) −3957 −3613 −3805 −3144 −2046 2342−3118 −2720 40700% — 394 45 96 359 117 −369 −294 −249 — 291(L) −3782−3025 −3298 −2972 −2345 −1269 −1846 1565 40800% — 394 45 96 359 117 −369−294 −249 — 292(K) −1949 889 −603 −763 783 −1968 −2532 −1851 40900% —394 45 96 359 117 −369 −294 −249 — 293(D) −2782 −1139 2142 −1879 −2163−3473 −4024 −3155 41000% — 394 45 96 359 117 −369 −294 −249 — 294(I)−4862 −4869 −5086 −4473 −2627 2071 −4467 −3968 41100% — 394 45 96 359117 −369 −294 −249 — 295(Q) −2936 3817 −430 −2018 −2031 −3060 −3278−2908 41200% — 394 45 96 359 117 −369 −294 −249 — 296(S) −2288 724 −11771978 −96 −2508 −3076 −2340 41300% — 394 45 96 359 117 −369 −294 −249 —297(G) −4804 −5546 −5385 −4727 −4815 −5862 −4924 −5849 41400% — 394 4596 359 117 −369 −294 −249 — 298(E) −1991 −97 777 829 1488 −1999 −2559−1890 41500% — 394 45 96 359 117 −369 −294 −249 — 299(F) −4750 −3628−4209 −4078 −4237 −3546 −601 2917 41600% — 394 45 96 359 117 −369 −294−249 — 300(A) −3432 −3073 −3298 −2387 197 1100 −2812 −2447 41700% — 39445 96 359 117 −369 −294 −249 — 301(K) −3047 −758 2488 −458 −2152 −3194−3159 −2895 41800% — 394 45 96 359 117 −369 −294 −249 — 302(M) −1958 −66722 775 −833 −1982 −2545 −1864 41900% — 394 45 96 359 117 −369 −294 −249— 303(W) −4179 −3330 −3743 −3475 −2878 664 4754 −84 42000% — 394 45 96359 117 −369 −294 −249 — 304(I) −4713 −4488 −4769 −4301 −2597 2384 −3934−3596 42100% — 394 45 96 359 117 −369 −294 −249 — 305(L) −2325 −915−1349 767 −152 84 −1756 −1329 42200% — 394 45 96 359 117 −369 −294 −249— 306(E) −2893 −1339 −2626 −2045 −2402 −3767 −4362 −3401 42300% — 394 4596 359 117 −369 −294 −249 — 307(N) −2155 −482 165 −1022 −880 30 −19901446 42400% — 394 45 96 359 117 −369 −294 −249 — 308(Q) −1989 2135 1528−812 −868 −2019 −2566 −1895 42500% — 394 45 96 359 117 −369 −294 −249 —309(A) −2103 −346 1657 −953 306 −261 −2169 −1624 42600% — 394 45 96 359117 −369 −294 −249 — 310(G) −3167 −2550 −3185 −1986 −2162 −3006 −2814−1874 42700% — 394 45 96 359 117 −369 −294 −249 — 311(Y) −2267 1002 1577−1172 −888 −800 −1730 2446 42800% — 394 45 96 359 117 −369 −294 −249 —312(P) 3598 −1697 −2362 −1566 286 −2803 −3832 −3277 42900% — 394 45 96359 117 −369 −294 −249 — 313(K) −1569 232 698 −786 −759 −1358 −1637−1317 43000% — 394 45 96 359 117 −369 −294 −249 — 314(E) −1441 −4 −527−653 −814 −1512 −1988 −1505 43100% — 394 45 96 359 117 −369 −294 −249 —315(T) −2004 −128 839 1365 1730 −2017 −2592 −1915 43200% — 394 45 96 359117 −369 −294 −249 — 316(M) −3673 −2938 −3257 −2944 −2114 326 −2014 166243300% — 394 45 96 359 117 −369 −294 −249 — 317(H) 472 −69 433 −772 1411−299 −2507 −1836 43400% — 394 45 96 359 117 −369 −294 −249 — 318(A)−2110 837 −805 −138 −1006 −2092 −2699 −2039 43500% — 394 45 96 359 117−369 −294 −249 — 319(M) −2474 −1419 1020 −1455 670 −411 831 535 43600% —394 45 96 359 117 −369 −294 −249 — 320(R) 597 −382 2908 −1246 −1219−2044 −2579 −2061 43700% — 394 45 96 359 117 −369 −294 −249 — 321(R)−2020 −177 2045 95 −893 −1984 −2543 −1892 43800% — 394 45 96 359 117−369 −294 −249 — 322(N) −1957 348 224 551 −821 −1910 −2497 −1828 43900%— 394 45 96 359 117 −369 −294 −249 — 323(E) −1963 682 −629 −781 12 −279−2472 476 44000% — 394 45 96 359 117 −369 −294 −249 — 324(N) −1827 1398447 −748 −766 −1713 −1779 −1595 44100% — 394 45 96 359 117 −369 −294−249 — 325(N) −2417 −657 −1430 −1361 197 −2727 −3303 −2540 44200% — 39445 96 359 117 −369 −294 −249 — 326(H) −2961 1288 −1287 670 −2133 −3161−3310 −2601 44300% — 394 45 96 359 117 −369 −294 −249 — 327(Q) 1404 2021−634 −785 619 −561 −2495 −1830 44400% — 394 45 96 359 117 −369 −294 −249— 328(I) −3016 −1615 822 −2097 −1556 −560 −2157 −1805 44500% — 394 45 96359 117 −369 −294 −249 — 329(E) −3440 −2054 −2556 −2649 −2820 −3234−3920 −3370 44600% — 394 45 96 359 117 −369 −294 −249 — 330(W) −2104−1454 −1405 −1757 −1583 −1218 5462 838 44700% — 394 45 96 359 117 −369−294 −249 — 331(K) −2030 1425 −709 −864 −350 1337 −2323 −1722 44800% —394 45 96 359 117 −369 −294 −249 — 332(V) −4575 −4456 −4653 −3990 11173227 −4065 −3597 44900% — 394 45 96 359 117 −369 −294 −249 — 333(G)−4804 −5546 −5385 −4727 −4815 −5862 −4924 −5849 45000% — 394 45 96 359117 −369 −294 −249 — 334(E) −2297 −451 1346 −1191 −1280 −2412 −2952−2288 45100% — 394 45 96 359 117 −369 −294 −249 — 335(K) −2200 1198 862−1073 −1133 −2287 −2770 −2133 45200% — 394 45 96 359 117 −369 −294 −249— 336(L) −4852 −4044 −4689 −4963 −3436 742 −3082 −3239 45300% — 394 4596 359 117 −369 −294 −249 — 337(R) −4754 −3672 4219 −4989 −4832 −5644−4538 −4993 45400% — 394 45 96 359 117 −369 −294 −249 — 338(E) −1998−117 −681 1129 −534 −2044 −2609 −1921 45500% — 394 45 96 359 117 −369−294 −249 — 339(M) −4742 −3963 −4548 −4689 −3273 771 −3038 −3137 45600%— 394 45 96 359 117 −369 −294 −249 — 340(M) −3334 −2559 641 −2503 −1740404 −1934 −1661 45700% — 394 45 96 359 117 −369 −294 −249 — 341(P) 3205695 −1607 703 −1207 −1700 −2613 −2121 45800% — 394 45 96 359 117 −369−294 −249 — 342(W) −4206 −2971 −3005 −3484 −3396 −2818 5644 611 45900% —394 45 96 359 117 −369 −294 −249 — 343(I) −4407 −4340 −4559 −3974 −22162150 −3915 −3445 46000% — 394 45 96 359 117 −369 −294 −249 — 344(A)−1822 37 −536 966 −724 −1859 −2439 −1755 46100% — 394 45 96 359 117 −369−294 −249 — 345(A) −1922 1335 −611 251 −768 −123 −2361 −1732 46200% —394 45 96 359 117 −369 −294 −249 — 346(N) 1624 −733 −1607 −1438 −1645−2887 −3466 −2662 46300% — 394 45 96 359 117 −369 −294 −249 — 347(K)−2774 −488 1175 −1994 −1888 −2848 −2822 −2610 46400% — 394 45 96 359 117−369 −294 −249 — 348(L) −2134 −805 485 −1085 −677 471 −1340 −944 46500%— 394 45 96 359 117 −369 −294 −249 — 349(V) −2830 −2012 −2236 −1892−1154 2537 −1701 −1344 46600% — 394 45 96 359 117 −369 −294 −249 —350(D) −2733 −1154 −2294 −1833 −2125 575 −3993 −3117 46700% — 394 45 96359 117 −369 −294 −249 — 351(K) 531 720 −562 −724 290 −1929 −2493 −181246800% — 394 45 96 359 117 −369 −294 −249 — 352(D) 34 −182 −708 541 −74−281 −2188 −1605 46900% — 394 45 96 359 117 −369 −294 −249 — 353(K)−2548 −407 1469 −1628 −1517 −119 −2497 1619 47000% — 394 45 96 359 117−369 −294 −249 — 354(N) 1327 −1460 −2119 1182 −1362 −2558 −3601 −304647100% — * * * * * * * * — HMMER2.0 [2.2 g] File format version: aunique identifier for this save file format. NAME Functionally VerifiedKARIs Name of the profile HMM LENG 354 Model length: the number of matchstates in the model. ALPH Amino Symbol alphbet: This determines thesymbol alphabet and the size of the symbol emission probabilitydistributions. IAmino, the alphabet size is set to 20 and the symbolalphabet to “ACDEFGHIKLMNPQRSTVWY” (alphabetic order). MAP yes Mapannotation flag: If set to yes, each line of data for the matchstate/consensus column in the main section of the file is followed by anextra number. This number gives the index of the alignment column thatthe match state was made from. This information provides a “map” of thematch states (1 . . . M) onto the columns of the alignment (1.alen). Itis used for quickly aligning the model back to the original alignment,e.g. when using hmmalign-mapali. COM hmmbuild-n Functionally Commandline for every HMMER command that modifies the save file: Verified KARIsexp-KARI.hmm This one means that hmmbuild (default patrameters) wasapplied to exp-KARI_mod.aln generate the save file. COM hmmcalibrateCommand line for every HMMER command that modifies the save file: Thisone exp-KARI.hmm means that hmmcalibrate (default parametrs) was appliedto the save profile. NSEQ 25 Sequence number: the number of sequencesthe HMM was trained on DATE Mon Dec 8 17:34:51 2008 Creation date: Whenwas the save file was generated. XT −8455 −4 −1000 −1000 Eight “special”transitions for controlling parts −8455 −4 −8455 −4 of thealgorithm-specific parts of the Plan7 model. The null probability usedto convert these back to model probabilities is 1.0. The order of theeight fields is N->B, N->N, E->C, E->J, C->T, C->C, J->B, J->J. NULT −4−8455 The transition probability distribution for the null model (singleG state). NULE 595 −1558 85 338 −294 453 The extreme value distributionparameters μ and lambda respectively; −1158 197 249 902 −1085 −142 bothfloating point values. These values are set when the model is calibrated−21 −313 45 531 201 384 −1998 −644 with hmmcalibrate. They are used todetermine E-values of bit scores. EVD −333.712708 0.110102

1. A mutant ketol-acid reductoisomerase enzyme comprising the amino acidsequence as set forth in SEQ ID NO:
 29. 2. A nucleic acid moleculeencoding the mutant ketol-acid reductoisomerase enzyme of claim
 1. 3. Anucleic acid molecule encoding a mutant ketol-acid reductoisomeraseenzyme have the nucleic acid sequence as set forth in SEQ ID NO:23.
 4. Amutant ketol-acid reductoisomerase enzyme encoded by the nucleic acidmolecule of claim
 3. 5. A recombinant cell comprising the mutantketol-acid reductoisomerase enzyme of claim
 1. 6. A mutant ketol-acidreductoisomerase enzyme as set forth in SEQ ID NO:17 comprising at leastone mutation at a residue selected from the group consisting of 24, 33,47, 50, 52, 53, 61, 80, 115, 156, 165, and
 170. 7. A mutant ketol-acidreductoisomerase enzyme according to claim 6 wherein: a) the residue atposition 47 has an amino acid substation selected from the groupconsisting of A, C, D, F, G, I, L, N, P, and Y; b) the residue atposition 50 has an amino acid substitution selected from the groupconsisting of A, C, D, E, F, G, M, N, V, W; c) the residue at position52 has an amino acid substitution selected from the group consisting ofA, C, D, G, H, N, S; d) the residue at position 53 has an amino acidsubstitution selected from the group consisting of A, H, I, W; e) theresidue at position 156 has an amino acid substitution of V; f) theresidue at position 165 has an amino acid substitution of M; g) theresidue at position 61 has an amino acid substitution of F; h) theresidue at position 170 has an amino acid substitution of A; i) theresidue at position 24 has an amino acid substitution of F; j) theresidue at position 33 has an amino acid substitution of L; k) theresidue at position 80 has an amino acid substitution of I; and l) theresidue at position 115 has an amino acid substitution of L.
 8. Anucleic acid molecule encoding the mutant ketol-acid reductoisomeraseenzyme of claim
 6. 9. A method for the evolution of a NADPH bindingketol-acid reductoisomerase enzyme to an NADH using form comprising: a)providing a ketol-acid reductoisomerase enzyme which uses NADPH having aspecific native amino acid sequence; b) identifying the cofactorswitching residues in the enzyme of a) based on the amino acid sequenceof the Pseudomonas fluorescens ketol-acid reductoisomerase enzyme as setfor the in SEQ ID NO:17 wherein the cofactor switching residues are atpositions selected from the group consisting of; 24, 33, 47, 50, 52, 53,61, 80, 115, 156, 165, and
 170. c) creating mutations in at least one ofthe cofactor switching residues of b) to create a mutant enzyme whereinsaid mutant enzyme binds NADH.
 10. The method of claim 9 wherein: a) theresidue at position 47 has an amino acid substation selected from thegroup consisting of A, C, D, F, G, I, L, N, P, and Y; b) the residue atposition 50 has an amino acid substitution selected from the groupconsisting of A, C, D, E, F, G, M, N, V, W; c) the residue at position52 has an amino acid substitution selected from the group consisting ofA, C, D, G, H, N, S; d) the residue at position 53 has an amino acidsubstitution selected from the group consisting of A, H, I, W; e) theresidue at position 156 has an amino acid substitution of V; f) theresidue at position 165 has an amino acid substitution of M; g) theresidue at position 61 has an amino acid substitution of F; h) theresidue at position 170 has an amino acid substitution of A; i) theresidue at position 24 has an amino acid substitution of F; j) theresidue at position 33 has an amino acid substitution of L; k) theresidue at position 80 has an amino acid substitution of I; and l) theresidue at position 115 has an amino acid substitution of L.
 11. Themethod of claim 9 wherein the ketol-acid reductoisomerase enzyme has theamino acid sequence as set forth in SEQ ID NO:
 29. 12. A method for theproduction of isobutanol comprising: a) providing a recombinantmicrobial host cell comprising the following genetic constructs: i) atleast one genetic construct encoding an acetolactate synthase enzyme forthe conversion of pyruvate to acetolactate; ii) at least one geneticconstruct encoding a ketol-acid reductoisomerase enzyme of either ofclaims 1 or 6; iii) at least one genetic construct encoding anacetohydroxy acid dehydratase for the conversion of2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c); iv) atleast one genetic construct encoding a branched-chain keto aciddecarboxylase, of the conversion of α-ketoisovalerate toisobutyraldehyde, (pathway step d); v) at least one genetic constructencoding a branched-chain alcohol dehydrogenase for the conversion ofisobutyraldehyde to isobutanol (pathway step e); and b) growing the hostcell of (a) under conditions where iso-butanol is produced.
 13. A methodfor the evolution and identification of a NADPH binding ketol-acidreductoisomerase enzyme to an NADH using form comprising: a) providing aketol-acid reductoisomerase enzyme which uses NADPH having a specificnative amino acid sequence; b) identifying the amino acid residues inthe native amino acid sequence whose side chains are in close proximityto the adenosyl 2′-phosphate of NADPH as mutagenesis targets; c)creating a library of mutant ketol-acid reductoisomerase enzymes fromthe class I ketol-acid reductoisomerase enzyme of step (a), having atleast one mutation in at least one of the mutagenesis target sites ofstep (b); and d) screening the library of mutant ketol-acidreductoisomerase enzymes of step (c) to identify NADH binding mutantketol-acid reductoisomerase enzyme.
 14. A mutant ketol-acidreductoisomerase enzyme having the amino acid sequence selected from thegroup consisting of SEQ ID NO: 19, 24, 25, 26, 27, 28, 67, 68, 69, and70.